Human 4-1BB receptor splicing variant

ABSTRACT

The invention relates to h4-1BBSV receptor polypeptides, polynucleotides encoding the polypeptides, methods for producing the polypeptides, in particular by expressing the polynucleotides, and agonists and antagonists of the polypeptides. The invention further relates to methods for utilizing such polynucleotides, polypeptides, agonists and antagonists for applications, which relate, in part, to research, diagnostic and clinical arts.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of and claims priority under35 U.S.C. §120 to U.S. application Ser. No. 09/739,394, filed Dec. 19,2000, which is a continuation of and claims priority under 35 U.S.C.§120 to U.S. application Ser. No. 09/253,549, filed Feb. 22, 1999, whichis a divisional of and claims priority under 35 U.S.C. §120 to U.S.application Ser. No. 08/816,605, filed Mar. 13, 1997 (now U.S. Pat. No.5,874,240, issued Feb. 23, 1999), which is a non-provisional of andclaims benefit under 35 U.S.C. §119(e) of Provisional Application No.60/013,474, filed Mar. 15, 1996, all of which are hereby incorporated byreference.

FIELD OF THE INVENTION

[0002] This invention relates, in part, to newly identifiedpolynucleotides and polypeptides; variants and derivatives of thepolynucleotides and polypeptides; processes for making thepolynucleotides and the polypeptides, and their variants andderivatives; agonists and antagonists of the polypeptides; and uses ofthe polynucleotides, polypeptides, variants, derivatives, agonists andantagonists. In particular, in these and in other regards, the inventionrelates to polynucleotides and polypeptides of human 4-1BB receptorsplicing variant, sometimes hereinafter referred to as “h4-1BBSVreceptor”.

BACKGROUND OF THE INVENTION

[0003] Many biological actions, for instance, response to certainstimuli and natural biological processes, are controlled by factors,such as cytokines. Many cytokines act through receptors by engaging thereceptor and producing an intra-cellular response.

[0004] For example, tumor necrosis factor (TNF), both alpha and beta,are cytokines which act through TNF receptors to regulate numerousbiological processes, including protection against infection andinduction of shock and inflammatory disease. The TNF molecules belong tothe “TNF-ligand” superfamily, and act together with their receptors orcounter-ligands, the “TNF-receptor” superfamily. So far, nine members ofthe TNF ligand superfamily have been identified and ten members of theTNF-receptor superfamily have been characterized.

[0005] Among the ligands there are included TNF-α, lymphotoxin-α(LT-α,also known as TNF-β), LT-β (found in complex heterotrimer LT-α2-β),FasL, CD40, CD27, CD30, 4-1BB, OX40 and nerve growth factor (NGF). Thesuperfamily of TNF receptors includes the p55TNF receptor, p75TNFreceptor, TNF receptor-related protein, FAS antigen or APL-1, CD40,CD27, CD30, 4-1BB, OX40, low affinity p75 and NGF-receptor (Meager, A.,Biologicals, 22:291-295 (1994).

[0006] All members of the TNF-ligand superfamily are expressed byactivated T-cells, implying that they are necessary for T-cellinteractions with other cell types which underlie cell ontogeny andfunctions. (Meager, A., supra).

[0007] Considerable insight into the essential functions of severalmembers of the TNF receptor family has been gained from theidentification and creation of mutants that abolish the expression ofthese proteins. For example, naturally occurring mutations in the FASantigen and its ligand cause lymphoproliferative disease(Watanabe-Fukunaga, R., et al., Nature, 356:314 (1992), perhapsreflecting a failure of programmed cell death. Mutations of the CD40ligand cause an X-linked immunodeficiency state characterized by highlevels of immunoglobulin M and low levels of immunoglobulin G in plasma,indicating faulty T-cell-dependent B-cell activation (Allen, R. C., etal., Science, 259:990 (1993). Targeted mutations of the low affinitynerve growth factor receptor cause a disorder characterized by faultysensory innovation of peripheral structures (Lee, K. F., et al., Cell,69:737 (1992).

[0008] TNF and LT-α are capable of binding to two TNF receptors (the 55-and 75-kd TNF receptors). A large number of biological effects elicitedby TNF and LT-α, acting through their receptors, include hemorrhagicnecrosis of transplanted tumors, cytotoxicity, a role in endotoxicshock, inflammation, immunoregulation, proliferation and anti-viralresponses, as well as protection against the deleterious effects ofionizing radiation. TNF and LT-α are involved in the pathogenesis of awide range of diseases, including endotoxic shock, cerebral malaria,tumors, autoimmune disease, AIDS and graft-host rejection (Beutler, B.and Von Huffel, C., Science, 264:667, 668 (1994). Mutations in the p55Receptor cause increased susceptibility to microbial infection.

[0009] 4-1BB ligand, a member of the TNF family of ligands, is inducedby T-cell activation. Signaling through a 4-1BB receptor enhancesproliferative T-cell responses. Among known 4-1BB receptors is theinducible murine T-cell 4-1BB receptor which is a member of the TNFreceptor family. It is expressed on activated T-cells as both a 30-kDamonomer and a 55-kDa homodimer (Pollok, K. E., et al., J. Immunol.,150:771 (1993). The 4-1BB receptor binds 4-1BB ligand with a highaffinity, and has been identified and cloned (Goodwin, R. G., et al.,Eur. J. Immunol., 23:2631 (1993). 4-1BB ligand was highly expressed onmature B and macrophage cell lines and anti-micro-activated B-cells.Recently, the human homolog of the murine 4-1BB receptor and its ligandhave been cloned (Schwarz, H. J., et al., Gene, 134:295 (1993). Datasuggests a potential role for the interaction of 4-1BB receptor with itsligand in the process of T-cell activation.

[0010] A gene has also been recently identified which is induced bylymphocyte activation. The sequence of the full length 1.4 kb cDNA hasbeen characterized as a new member of the nerve growth factor/tumornecrosis factor receptor family and is considered to be the humanhomolog of the murine T-cell-specific receptor 4-1BB. This receptor genecan be induced in lymphoid and differentiated non-lymphoid cell types.Expression of the protein encoded by this gene has been found on asubset of activated T or B lymphocytes. Activation-dependent expressionof the protein is found not only in T lymphocytes, but also in Blymphocytes, monocytes and diverse non-lymphoid cell types (Schwarz,Blood, 85 (4):1043-1052 (1995).

[0011] The effects of TNF family ligands and TNF family receptors arevaried and influence numerous functions, both normal and abnormal, inthe biological processes of the mammalian system. There is a clear need,therefore, for identification and characterization of such receptors andligands that influence biological activity, both normally and in diseasestates. In particular, there is a need to isolate and characterizeadditional NGF/TNF family receptors akin to 4-1BB which enhancesproliferative T-cell responses and may be employed, therefore, forpreventing, ameliorating or correcting dysfunctions or disease oraugmenting positive natural actions of such receptors.

SUMMARY OF THE INVENTION

[0012] Toward these ends, and others, it is an object of the presentinvention to provide receptor polypeptides, inter alia, that have beenidentified as novel h4-1BBSV receptors by homology between the aminoacid sequence set out in FIGS. 1A-B (SEQ ID NO:2) and known amino acidsequences of other proteins such as the human 4-1BB receptor protein(SEQ ID NO:9) which may have a different tissue distribution and adifferent specificity for 4-1BB ligand.

[0013] It is a further object of the invention, moreover, to providepolynucleotides that encode h4-1BBSV receptors, particularlypolynucleotides that encode the polypeptide herein designated h4-1BBSVreceptor.

[0014] In a particularly preferred embodiment of this aspect of theinvention the polynucleotide comprises the region encoding h4-1BBSVreceptor in the sequence set out in FIGS. 1A-B (SEQ ID NO:2).

[0015] In accordance with this aspect of the present invention there isprovided an isolated nucleic acid molecule encoding a mature polypeptideexpressed by the human cDNA contained in the deposited clone which ishereinafter described.

[0016] In accordance with this aspect of the invention there areprovided isolated nucleic acid molecules encoding h4-1BBSV receptors,including mRNAs, cDNAs, genomic DNAs and, in further embodiments of thisaspect of the invention, biologically, diagnostically, clinically ortherapeutically useful variants, analogs or derivatives thereof, orfragments thereof, including fragments of the variants, analogs andderivatives.

[0017] Among the particularly preferred embodiments of this aspect ofthe invention are naturally occurring allelic variants of h4-1BBSVreceptor.

[0018] In accordance with this aspect of the invention there areprovided novel receptor polypeptides of human origin referred to hereinas h4-1BBSV as well as biologically, diagnostically or therapeuticallyuseful fragments, variants and derivatives thereof, variants andderivatives of the fragments, and analogs of the foregoing.

[0019] Among the particularly preferred embodiments of this aspect ofthe invention are variants of h4-1BBSV receptor encoded by naturallyoccurring alleles of the h4-1BBSV receptor gene.

[0020] It is another object of the invention to provide a process forproducing the aforementioned polypeptides, polypeptide fragments,variants and derivatives, fragments of the variants and derivatives, andanalogs of the foregoing.

[0021] In a preferred embodiment of this aspect of the invention thereare provided methods for producing the aforementioned h4-1BBSV receptorpolypeptides comprising culturing host cells having expressiblyincorporated therein an exogenously-derived h4-1BBSV-encodingpolynucleotide under conditions for expression of h4-1BBSV receptor inthe host and then recovering the expressed polypeptide.

[0022] In accordance with another object the invention there areprovided products, compositions, processes and methods that utilize theaforementioned polypeptides and polynucleotides for research,biological, clinical and therapeutic purposes, inter alia.

[0023] In accordance with certain preferred embodiments of this aspectof the invention, there are provided products, compositions and methods,inter alia, for, among other things: assessing h4-1BBSV expression incells by determining h4-1BBSV polypeptides or h4-1BBSV-encoding mRNA;assaying genetic variation and aberrations, such as defects, in h4-1BBSVgenes; and administering a h4-1BBSV polypeptide or polynucleotide to anorganism to augment h4-1BBSV function or remediate h4-1BBSV dysfunction.

[0024] In accordance with certain preferred embodiments of this andother aspects of the invention there are provided probes that hybridizeto human h4-1BBSV sequences.

[0025] In certain additional preferred embodiments of this aspect of theinvention there are provided antibodies against h4-1BBSV polypeptides.In certain particularly preferred embodiments in this regard, theantibodies are highly selective for h4-1BBSV.

[0026] In accordance with another aspect of the present invention, thereare provided h4-1BBSV agonists. Among preferred agonists are moleculesthat bind to h4-1BBSV receptor molecules, and that elicit or augmenth4-1BBSV receptor responses. Also among preferred agonists are moleculesthat interact with h4-1BBSV receptor polypeptides, or with othermodulators of h4-1BBSV receptor activities, and thereby potentiate oraugment an effect of h4-1BBSV receptor or more than one effect ofh4-1BBSV receptor, for example, the agonists may be employed to treatand or prevent tumors, cytotoxicity, viral infection, deleteriouseffects of ionizing radiation, autoimmune disease, AIDS and graft-hostrejection, to regulate immune responses, and cellular proliferation.

[0027] In accordance with yet another aspect of the present invention,there are provided h4-1BBSV antagonists. Among preferred antagonists arethose which bind to h4-1BBSV receptor or binding molecules and do notelicit a h4-1BBSV receptor response or more than one h4-1BBSV receptorresponse. Also among preferred antagonists are soluble forms of theh4-1BBSV receptor which bind to or interact with ligands thereof so asto inhibit an effect of h4-1BBSV receptor or more than one effect ofh4-1BBSV receptor or which prevent expression h4-1BBSV.

[0028] The antagonists may be used to inhibit the action of h4-1BBSVreceptor polypeptides. They may be used, for instance, to treat and/orprevent endotoxic shock, inflammation, cerebral malaria, activation ofthe HIV virus, graft rejection, bone resorption and cachexia.

[0029] In a further aspect of the invention there are providedcompositions comprising a h4-1BBSV receptor polynucleotide or a h4-1BBSVreceptor polypeptide for administration to cells in vitro, to cells exvivo and to cells in vivo, or to a multicellular organism. In certainparticularly preferred embodiments of this aspect of the invention, thecompositions comprise a h4-1BBSV receptor polynucleotide for expressionof a h4-1BBSV receptor polypeptide in a host organism for treatment ofdisease. Particularly preferred in this regard is expression in a humanpatient for treatment of a dysfunction associated with aberrantendogenous activity of h4-1BBSV receptor.

[0030] Other objects, features, advantages and aspects of the presentinvention will become apparent to those of skill from the followingdescription. It should be understood, however, that the followingdescription and the specific examples, while indicating preferredembodiments of the invention, are given by way of illustration only.Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following description and from reading the otherparts of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The following drawings depict certain embodiments of theinvention. They are illustrative only and do not limit the inventionotherwise disclosed herein.

[0032] FIGS. 1A-B show the nucleotide (SEQ ID NO:1) and deduced aminoacid (SEQ ID NO:2) sequences of the h4-1BBSV receptor.

[0033]FIG. 2 shows the regions of similarity between amino acidsequences of h4-1BBSV receptor (SEQ ID NO:2) and human 4-1BB receptorpolypeptide as shown in Schwarz, H. J., et al., Gene, 134:295 (1993)(SEQID NO:9).

[0034]FIG. 3 shows structural and functional features of h4-1BBSVreceptor deduced by the indicated techniques, as a function of aminoacid sequence.

GLOSSARY

[0035] The following illustrative explanations are provided tofacilitate understanding of certain terms used frequently herein,particularly in the examples. The explanations are provided as aconvenience and are not limitative of the invention.

[0036] DIGESTION of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes referred to herein are commerciallyavailable and their reaction conditions, cofactors and otherrequirements for use are known and routine to the skilled artisan.

[0037] For analytical purposes, typically, 1 μg of plasmid or DNAfragment is digested with about 2 units of enzyme in about 20 μl ofreaction buffer. For the purpose of isolating DNA fragments for plasmidconstruction, typically 5 to 50 μg of DNA are digested with 20 to 250units of enzyme in proportionately larger volumes.

[0038] Appropriate buffers and substrate amounts for particularrestriction enzymes are described in standard laboratory manuals, suchas those referenced below, and they are specified by commercialsuppliers.

[0039] Incubation times of about 1 hour at 37° C. are ordinarily used,but conditions may vary in accordance with standard procedures, thesupplier's instructions and the particulars of the reaction. Afterdigestion, reactions may be analyzed, and fragments may be purified byelectrophoresis through an agarose or polyacrylamide gel, using wellknown methods that are routine for those skilled in the art.

[0040] GENETIC ELEMENT generally means a polynucleotide comprising aregion that encodes a polypeptide or a region that regulatestranscription or translation or other processes important to expressionof the polypeptide in a host cell, or a polynucleotide comprising both aregion that encodes a polypeptide and a region operably linked theretothat regulates expression.

[0041] Genetic elements may be comprised within a vector that replicatesas an episomal element; that is, as a molecule physically independent ofthe host cell genome. They may be comprised within mini-chromosomes,such as those that arise during amplification of transfected DNA bymethotrexate selection in eukaryotic cells. Genetic elements also may becomprised within a host cell genome; not in their natural state but,rather, following manipulation such as isolation, cloning andintroduction into a host cell in the form of purified DNA or in avector, among others.

[0042] ISOLATED means altered “by the hand of man” from its naturalstate; i.e., that, if it occurs in nature, it has been changed orremoved from its original environment, or both.

[0043] For example, a naturally occurring polynucleotide or apolypeptide naturally present in a living animal in its natural state isnot “isolated,” but the same polynucleotide or polypeptide separatedfrom the coexisting materials of its natural state is “isolated”, as theterm is employed herein. For example, with respect to polynucleotides,the term isolated means that it is separated from the chromosome andcell in which it naturally occurs.

[0044] As part of or following isolation, such polynucleotides can bejoined to other polynucleotides, such as DNAs, for mutagenesis, to formfusion proteins, and for propagation or expression in a host, forinstance. The isolated polynucleotides, alone or joined to otherpolynucleotides such as vectors, can be introduced into host cells, inculture or in whole organisms. Introduced into host cells in culture orin whole organisms, such DNAs still would be isolated, as the term isused herein, because they would not be in their naturally occurring formor environment. Similarly, the polynucleotides and polypeptides mayoccur in a composition, such as a media formulations, solutions forintroduction of polynucleotides or polypeptides, for example, intocells, compositions or solutions for chemical or enzymatic reactions,for instance, which are not naturally occurring compositions, and,therein remain isolated polynucleotides or polypeptides within themeaning of that term as it is employed herein.

[0045] LIGATION refers to the process of forming phosphodiester bondsbetween two or more polynucleotides, which most often are doublestranded DNAs. Techniques for ligation are well known to the art andprotocols for ligation are described in standard laboratory manuals andreferences, such as, for instance, Sambrook et al., MOLECULAR CLONING, ALABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989) and Maniatis et al., pg. 146, as cited below.

[0046] OLIGONUCLEOTIDE(S) refers to relatively short polynucleotides.Often the term refers to single-stranded deoxyribonucleotide, but it canrefer as well to single-or double-stranded ribonucleotide, RNA:DNAhybrids and double-stranded DNAS, among others.

[0047] Oligonucleotides, such as single-stranded DNA probeoligonucleotides, often are synthesized by chemical methods, such asthose implemented on automated oligonucleotide synthesizers. However,oligonucleotides can be made by a variety of other methods, including invitro recombinant DNA-mediated techniques and by expression of DNAs incells and organisms.

[0048] Initially, chemically synthesized DNAs typically are obtainedwithout a 5′ phosphate. The 5′ ends of such oligonucleotides are notsubstrates for phosphodiester bond formation by ligation reactions thatemploy DNA ligase typically used to form recombinant DNA molecules.Where ligation of such oligonucleotides is desired, a phosphate can beadded by standard techniques, such as those that employ a kinase andATP.

[0049] The 3′ end of a chemically synthesized oligonucleotide generallyhas a free hydroxyl group and, in the presence of a ligase, such as T4DNA ligase, readily will form a phosphodiester bond with a 5′ phosphateof another polynucleotide, such as another oligonucleotide. As is wellknown, this reaction can be prevented selectively, where desired, byremoving the 5′ phosphates of the other polynucleotide(s) prior toligation.

[0050] PLASMIDS generally are designated herein by a lower case ppreceded and/or followed by capital letters and/or numbers, inaccordance with standard naming conventions that are familiar to thoseof skill in the art. Starting plasmids disclosed herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids by routine application ofwell known, published procedures. Many plasmids and other cloning andexpression vectors that can be used in accordance with the presentinvention are well known and readily available to those of skill in theart. Moreover, those of skill readily may construct any number of otherplasmids suitable for use in the invention. The properties, constructionand use of such plasmids, as well as other vectors, in the presentinvention will be readily apparent to those of skill from the presentdisclosure.

[0051] POLYNUCLEOTIDE(S) generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. Thus, for instance, polynucleotides as used herein refersto, among others, single-and double-stranded DNA, DNA that is a mixtureof single-and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions.

[0052] In addition, polynucleotide as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.

[0053] As used herein, the term polynucleotide includes DNAs or RNAs asdescribed above that contain one or more modified bases. Thus, DNAs orRNAs with backbones modified for stability or for other reasons are“polynucleotides” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein.

[0054] It will be appreciated that a great variety of modifications havebeen made to DNA and RNA that serve many useful purposes known to thoseof skill in the art. The term polynucleotide as it is employed hereinembraces such chemically, enzymatically or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including simple and complex cells,inter alia.

[0055] POLYPEPTIDES, as used herein, includes all polypeptides asdescribed below. The basic structure of polypeptides is well known andhas been described in innumerable textbooks and other publications inthe art. In this context, the term is used herein to refer to anypeptide or protein comprising two or more amino acids joined to eachother in a linear chain by peptide bonds. As used herein, the termrefers to both short chains, which also commonly are referred to in theart as peptides, oligopeptides and oligomers, for example, and to longerchains, which generally are referred to in the art as proteins, of whichthere are many types.

[0056] It will be appreciated that polypeptides often contain aminoacids other than the 20 amino acids commonly referred to as the 20naturally occurring amino acids, and that many amino acids, includingthe terminal amino acids, may be modified in a given polypeptide, eitherby natural processes, such as processing and other post-translationalmodifications, but also by chemical modification techniques which arewell known to the art. Even the common modifications that occurnaturally in polypeptides are too numerous to list exhaustively here,but they are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature, and they arewell known to those of skill in the art. Among the known modificationswhich may be present in polypeptides of the present are, to name anillustrative few, acetylation, acylation, ADP-ribosylation, amidation,covalent attachment of flavin, covalent attachment of a heme moiety,covalent attachment of a nucleotide or nucleotide derivative, covalentattachment of a lipid or lipid derivative, covalent attachment ofphosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutamate, formulation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, selenoylation,sulfation, transfer-RNA mediated addition of amino acids to proteinssuch as arginylation, and ubiquitination.

[0057] Such modifications are well known to those of skill and have beendescribed in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as, for instance PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993). Manydetailed reviews are available on this subject, such as, for example,those provided by Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York(1983); Seifter et al., Analysis for protein modifications andnonprotein cofactors, Meth. Enzymol. 182: 626-646 (1990) and Rattan etal., Protein Synthesis: Posttranslational Modifications and Aging, Ann.N.Y. Acad. Sci. 663: 48-62 (1992).

[0058] In general, as used herein, the term polypeptide encompasses allsuch modifications, particularly those that are present in polypeptidessynthesized by expressing a polynucleotide in a host cell.

[0059] VARIANT(S) of polynucleotides or polypeptides, as the term isused herein, are polynucleotides or polypeptides that differ from areference polynucleotide or polypeptide, respectively. Variants in thissense are described below and elsewhere in the present disclosure ingreater detail.

[0060] A polynucleotide that differs in nucleotide sequence fromanother, reference polynucleotide. Generally, differences are limited sothat the nucleotide sequences of the reference and the variant areclosely similar overall and, in many regions, identical.

[0061] As noted below, changes in the nucleotide sequence of the variantmay be silent. That is, they may not alter the amino acids encoded bythe polynucleotide. Where alterations are limited to silent changes ofthis type a variant will encode a polypeptide with the same amino acidsequence as the reference. Also as noted below, changes in thenucleotide sequence of the variant may alter the amino acid sequence ofa polypeptide encoded by the reference polynucleotide. Such nucleotidechanges may result in amino acid substitutions, additions, deletions,fusions and truncations in the polypeptide encoded by the referencesequence, as discussed below.

[0062] A polypeptide that differs in amino acid sequence from another,reference polypeptide. Generally, differences are limited so that thesequences of the reference and the variant are closely similar overalland, in many region, identical.

[0063] A variant and reference polypeptide may differ in amino acidsequence by one or more substitutions, additions, deletions, fusions andtruncations, which may be present in any combination.

DESCRIPTION OF THE INVENTION

[0064] The present invention relates to novel h4-1BBSV receptorpolypeptides and polynucleotides, among other things, as described ingreater detail below. In particular, the invention relates topolypeptides and polynucleotides of a novel h4-1BBSV receptor, which isrelated by amino acid sequence homology to human h4-1BB receptorpolypeptide (SEQ ID NO:9). The invention relates especially to h4-1BBSVreceptor having the nucleotide and amino acid sequences set out in FIGS.1A-B (SEQ ID NO:1-2), and to the h4-1BBSV receptor nucleotide and aminoacid sequences of the human cDNA in FIGS. 1A-B (SEQ ID NO:1) obtained bysequencing the cDNA of the deposited clone, hereinafter described.Hence, the sequence of the deposited clone is controlling as to anydiscrepancies between the two and any reference to the sequences ofFIGS. 1A-B (SEQ ID NO:1) include reference to the sequence of the humancDNA of the deposited clone. Polynucleotides

[0065] In accordance with one aspect of the present invention, there areprovided isolated polynucleotides which encode the h4-1BBSV receptorpolypeptide having the deduced amino acid sequence of FIGS. 1A-B (SEQ IDNO:2).

[0066] Using the information provided herein, such as the polynucleotidesequence set out in FIGS. 1A-B (SEQ ID NO:1), a polynucleotide of thepresent invention encoding h4-1BBSV receptor polypeptide may be obtainedusing standard cloning and screening procedures, such as those forcloning cDNAs using mRNA as starting material. Illustrative of theinvention, the polynucleotide set out in FIGS. 1A-B (SEQ ID NO:1) wasdiscovered in a cDNA library derived from cells of a human activatedT-cell (12 hours)/thiouridine labelled Eco.

[0067] The h4-1BBSV receptor of the invention is structurally related toother proteins of the NGF Receptor family, as shown by the results ofsequencing the human cDNA encoding h4-1BBSV receptor in the depositedclone, hereinafter described. The human cDNA sequence thus obtained isset out in FIGS. 1A-B (SEQ ID NO:1). It contains an open reading frameencoding a protein of about 219 amino acid residues with a deducedmolecular weight of about 24.1 kDa. The initial 18 amino acids representa putative leader sequence (residues −18 to −1 of SEQ ID NO:2) with thenext 132 amino acids being a soluble extracellular domain (residues 1 to132 of SEQ ID NO:2) and the next 27 amino acids being a transmembranedomain (residues 133 to 159 of SEQ ID NO:2). The protein exhibitsgreatest degree of homology to human 4-1BB receptor protein (SEQ IDNO:9) among known proteins. The h4-1BBSV receptor of FIGS. 1A-B (SEQ IDNO:2) does not contain the second exon of human 4-1BB receptor protein,which encodes thirty-six amino acids (residues 34-69 of SEQ ID NO:9);otherwise it is identical to the amino acid sequence of human 4-1BBreceptor protein (SEQ ID NO:9).

[0068] Polynucleotides of the present invention may be in the form ofRNA, such as mRNA, or in the form of DNA, including, for instance, cDNAand genomic DNA obtained by cloning or produced by chemical synthetictechniques or by a combination thereof. The DNA may be double-strandedor single-stranded. Single-stranded DNA may be the coding strand, alsoknown as the sense strand, or it may be the non-coding strand, alsoreferred to as the anti-sense strand.

[0069] The coding sequence which encodes the polypeptide may beidentical to the coding sequence of the polynucleotide shown in FIGS.1A-B (SEQ ID NO:1). It also may be a polynucleotide with a differentsequence, which, as a result of the redundancy (degeneracy) of thegenetic code, encodes the polypeptide of the DNA of FIGS. 1A-B (SEQ IDNO:2).

[0070] Polynucleotides of the present invention which encode thepolypeptide of FIGS. 1A-B (SEQ ID NO:2) may include, but are not limitedto the coding sequence for the mature polypeptide, by itself; the codingsequence for the mature polypeptide and additional coding sequences,such as those encoding a leader or secretory sequence, such as a pre-,or pro- or prepro-protein sequence; the coding sequence of the maturepolypeptide, with or without the aforementioned additional codingsequences, together with additional, non-coding sequences, including forexample, but not limited to introns and non-coding 5′ and 3′ sequences,such as the transcribed, non-translated sequences that play a role intranscription, mRNA processing—including splicing and polyadenylationsignals, for example—ribosome binding and stability of mRNA; additionalcoding sequence which codes for additional amino acids, such as thosewhich provide additional functionalities. Thus, for instance, thepolypeptide may be fused to a marker sequence, such as a peptide, whichfacilitates purification of the fused polypeptide. In certain preferredembodiments of this aspect of the invention, the marker sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(Qiagen, Inc.), among others, many of which are commercially available.As described in Gentz et al., Proc. Natl. Acad. Sci., USA 86: 821-824(1989), for instance, hexa-histidine provides for convenientpurification of the fusion protein.

[0071] The HA tag corresponds to an epitope derived of influenzahemagglutinin protein, which has been described by Wilson et al., Cell37: 767 (1984), for instance.

[0072] In accordance with the foregoing, the term “polynucleotideencoding a polypeptide” as used herein encompasses polynucleotides whichinclude a sequence encoding a polypeptide of the present invention,particularly h4-1BBSV receptor having the amino acid sequence set out inFIGS. 1A-B (SEQ ID NO:2). The term encompasses polynucleotides thatinclude a single continuous region or discontinuous regions encoding thepolypeptide (for example, interrupted by introns) together withadditional regions, that also may contain coding and/or non-codingsequences.

[0073] The present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs andderivatives of the polypeptide having the deduced amino acid sequence ofFIGS. 1A-B (SEQ ID NO:2). A variant of the polynucleotide may be anaturally occurring variant such as a naturally occurring allelicvariant, or it may be a variant that is not known to occur naturally.Such non-naturally occurring variants of the polynucleotide may be madeby mutagenesis techniques, including those applied to polynucleotides,cells or organisms.

[0074] Among variants in this regard are variants that differ from theaforementioned polynucleotides by nucleotide substitutions, deletions oradditions. The substitutions, deletions or additions may involve one ormore nucleotides. The variants may be altered in coding or non-codingregions or both. Alterations in the coding regions may produceconservative or non-conservative amino acid substitutions, deletions oradditions.

[0075] Among the particularly preferred embodiments of the invention inthis regard are polynucleotides encoding polypeptides having the aminoacid sequence of h4-1BBSV receptor set out in FIGS. 1A-B (SEQ ID NO:2);variants, analogs, derivatives and fragments thereof, and fragments ofthe variants, analogs and derivatives.

[0076] Further particularly preferred in this regard are polynucleotidesencoding h4-1BBSV receptor variants, analogs, derivatives and fragments,and variants, analogs and derivatives of the fragments, which have theamino acid sequence of the h4-1BBSV receptor polypeptide of FIGS. 1A-B(SEQ ID NO:2) in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 orno amino acid residues are substituted, deleted or added, in anycombination. Especially preferred among these are silent substitutions,additions and deletions, which do not alter the properties andactivities of the h4-1BBSV receptor. Also especially preferred in thisregard are conservative substitutions. Most highly preferred arepolynucleotides encoding polypeptides having the amino acid sequence ofFIGS. 1A-B (SEQ ID NO:2), without substitutions.

[0077] Further preferred embodiments of the invention arepolynucleotides that are at least 70% identical to a polynucleotideencoding the h4-1BBSV receptor polypeptide having the amino acidsequence set out in FIGS. 1A-B (SEQ ID NO:2), and polynucleotides whichare complementary to such polynucleotides. Alternatively, most highlypreferred are polynucleotides that comprise a region that is at least80% identical to a polynucleotide encoding the h4-1BBSV receptorpolypeptide of the human cDNA of the deposited clone, hereinafterdescribed, and polynucleotides complementary thereto. In this regard,polynucleotides at least 90% identical to the same are particularlypreferred, and among these particularly preferred polynucleotides, thosewith at least 95% are especially preferred. Furthermore, those with atleast 97% are highly preferred among those with at least 95%, and amongthese those with at least 98% and at least 99% are particularly highlypreferred, with at least 99% being the more preferred.

[0078] Particularly preferred embodiments in this respect, moreover, arepolynucleotides which encode polypeptides which retain substantially thesame biological function or activity as the mature polypeptide encodedby the human cDNA of FIGS. 1A-B (SEQ ID NO:1).

[0079] The present invention further relates to polynucleotides thathybridize to the herein above-described sequences. In this regard, thepresent invention especially relates to polynucleotides which hybridizeunder stringent conditions to the herein above-describedpolynucleotides. As herein used, the term “stringent conditions” meanshybridization will occur only if there is at least 95% and preferably atleast 97% identity between the sequences.

[0080] As discussed additionally herein regarding polynucleotide assaysof the invention, for instance, polynucleotides of the invention asdiscussed above, may be used as a hybridization probe for cDNA andgenomic DNA to isolate full-length cDNAs and genomic clones encodingh4-1BBSV receptor and to isolate cDNA and genomic clones of other genesthat have a high sequence similarity to the h4-1BBSV receptor gene. Suchprobes generally will comprise at least 15 bases. Preferably, suchprobes will have at least 30 bases and may have at least 50 bases.

[0081] For example, the coding region of the h4-1BBSV receptor gene maybe isolated by screening using the known DNA sequence to synthesize anoligonucleotide probe. A labeled oligonucleotide having a sequencecomplementary to that of a gene of the present invention is then used toscreen a library of human cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

[0082] The polynucleotides and polypeptides of the present invention maybe employed as research reagents and materials for discovery oftreatments and diagnostics to human disease, as further discussed hereinrelating to polynucleotide assays, inter alia.

[0083] The polynucleotides may encode a polypeptide which is the matureprotein plus additional amino or carboxyl-terminal amino acids, or aminoacids interior to the mature polypeptide (when the mature form has morethan one polypeptide chain, for instance). Such sequences may play arole in processing of a protein from precursor to a mature form, mayfacilitate protein trafficking, may prolong or shorten protein half-lifeor may facilitate manipulation of a protein for assay or production,among other things. As generally is the case in situ, the additionalamino acids may be processed away from the mature protein by cellularenzymes.

[0084] In sum, a polynucleotide of the present invention may encode amature protein, a mature protein plus a leader sequence (which may bereferred to as a preprotein), a precursor of a mature protein having oneor more prosequences which are not the leader sequences of a preprotein,or a preproprotein, which is a precursor to a proprotein, having aleader sequence and one or more prosequences, which generally areremoved during processing steps that produce active and mature forms ofthe polypeptide.

[0085] Deposited Materials

[0086] A deposit containing a h4-1BBSV receptor cDNA has been depositedwith the American Type Culture Collection, as noted above. The depositedclone was deposited with the American Type Culture Collection (“ATCC”),10801 University Blvd., Manassas, Va. 20110-2209, USA (present address),on Mar. 6, 1996 and assigned ATCC Deposit No. 97462.

[0087] The deposited clone is a pBluescript SK (−) plasmid (Stratagene,La Jolla, Calif.) that contains the full length h4-1BBSV receptor cDNA,referred to as “PF254” upon deposit.

[0088] The deposit has been made under the terms of the Budapest Treatyon the international recognition of the deposit of micro-organisms forpurposes of patent procedure. The strain will be irrevocably and withoutrestriction or condition released to the public upon the issuance of apatent. The deposit is provided merely as convenience to those of skillin the art and is not an admission that a deposit is required forenablement, such as that required under 35 U.S.C. §112.

[0089] The sequence of the polynucleotides contained in the depositedmaterial, as well as the amino acid sequence of the polypeptide encodedthereby, are controlling in the event of any conflict with anydescription of sequences herein.

[0090] A license may be required to make, use or sell the depositedmaterials, and no such license is hereby granted.

[0091] Polypeptides

[0092] The present invention further relates to a h4-1BBSV receptorpolypeptide which has the deduced amino acid sequence of FIGS. 1A-B (SEQID NO:2).

[0093] The invention also relates to fragments, analogs and derivativesof these polypeptides. The terms “fragment,” “derivative” and “analog”when referring to the polypeptide of FIGS. 1A-B (SEQ ID NO:2), means apolypeptide which retains essentially the same biological function oractivity as such polypeptide. Thus, an analog includes a proproteinwhich can be activated by cleavage of the proprotein portion to producean active mature polypeptide.

[0094] The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide. Incertain preferred embodiments it is a recombinant polypeptide.

[0095] Among the particularly preferred embodiments of the invention inthis regard are polypeptides having the amino acid sequence of h4-1BBSVreceptor set out in FIGS. 1A-B (SEQ ID NO:2), variants, analogs,derivatives and fragments thereof, and variants, analogs and derivativesof the fragments. Alternatively, particularly preferred embodiments ofthe invention in this regard are polypeptides having the amino acidsequence of the h4-1BBSV receptor of the cDNA in the deposited clone,variants, analogs, derivatives and fragments thereof, and variants,analogs and derivatives of the fragments.

[0096] Among preferred variants are those that vary from a reference byconservative amino acid substitutions. Such substitutions are those thatsubstitute a given amino acid in a polypeptide by another amino acid oflike characteristics. Typically seen as conservative substitutions arethe replacements, one for another, among the aliphatic amino acids Ala,Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe, Tyr.

[0097] Further particularly preferred in this regard are variants,analogs, derivatives and fragments, and variants, analogs andderivatives of the fragments, having the amino acid sequence of theh4-1BBSV receptor polypeptide of FIGS. 1A-B (SEQ ID NO:2), in whichseveral, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residuesare substituted, deleted or added, in any combination. Especiallypreferred among these are silent substitutions, additions and deletions,which do not alter the properties and activities of the h4-1BBSVreceptor. Also especially preferred in this regard are conservativesubstitutions. Most highly preferred are polypeptides having the aminoacid sequence of FIGS. 1A-B (SEQ ID NO:2) without substitutions.

[0098] The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

[0099] The polypeptides of the present invention include the polypeptideof SEQ ID NO:2 (in particular the mature polypeptide) as well aspolypeptides which have at least 70% similarity (preferably at least 70%identity) to the polypeptide of SEQ ID NO:2 and more preferably at least90% similarity (more preferably at least 90% identity) to thepolypeptide of SEQ ID NO:2 and still more preferably at least 95%similarity (still more preferably at least 95% identity) to thepolypeptide of SEQ ID NO:2 and also include portions of suchpolypeptides with such portion of the polypeptide generally containingat least 30 amino acids and more preferably at least 50 amino acids.

[0100] As known in the art “similarity” between two polypeptides isdetermined by comparing the amino acid sequence and its conserved aminoacid substitutes of one polypeptide to the sequence of a secondpolypeptide.

[0101] Fragments or portions of the polypeptides of the presentinvention may be employed for producing the corresponding full-lengthpolypeptide by peptide synthesis; therefore, the fragments may beemployed as intermediates for producing the full-length polypeptides.Fragments or portions of the polynucleotides of the present inventionmay be used to synthesize full-length polynucleotides of the presentinvention.

[0102] Fragments

[0103] Also among preferred embodiments of this aspect of the presentinvention are polypeptides comprising fragments of h4-1BBSV receptor,most particularly fragments of the h4-1BBSV receptor having the aminoacid set out in FIGS. 1A-B (SEQ ID NO:2), and fragments of variants andderivatives of the h4-1BBSV receptor of FIGS. 1A-B (SEQ ID NO:2).

[0104] In this regard a fragment is a polypeptide having an amino acidsequence that entirely is the same as part but not all of the amino acidsequence of the aforementioned h4-1BBSV receptor polypeptides andvariants or derivatives thereof.

[0105] Such fragments may be “free-standing,” i.e., not part of or fusedto other amino acids or polypeptides, or they may be comprised within alarger polypeptide of which they form a part or region. When comprisedwithin a larger polypeptide, the presently discussed fragments mostpreferably form a single continuous region. However, several fragmentsmay be comprised within a single larger polypeptide. For instance,certain preferred embodiments relate to a fragment of a h4-1BBSVreceptor polypeptide of the present comprised within a precursorpolypeptide designed for expression in a host and having heterologouspre and pro-polypeptide regions fused to the amino terminus of theh4-1BBSV receptor fragment and an additional region fused to thecarboxyl terminus of the fragment. Therefore, fragments in one aspect ofthe meaning intended herein, refers to the portion or portions of afusion polypeptide or fusion protein derived from h4-1BBSV receptor.

[0106] As representative examples of polypeptide fragments of theinvention, there may be mentioned those which have from about 150 toabout 219 amino acids.

[0107] In this context about includes the particularly recited range andranges larger or smaller by several, a few, 5, 4, 3, 2 or 1 amino acidat either extreme or at both extremes. For instance, about 150 to about219 amino acids in this context means a polypeptide fragment of 150 plusor minus several, a few, 5, 4, 3, 2 or 1 amino acids to 219 plus orminus several a few, 5, 4, 3, 2 or 1 amino acid residues, i.e., rangesas broad as 150 minus several amino acids to 219 plus several aminoacids to as narrow as 150 plus several amino acids to 219 minus severalamino acids.

[0108] Highly preferred in this regard are the recited ranges plus orminus as many as 5 amino acids at either or at both extremes.Particularly highly preferred are the recited ranges plus or minus asmany as 3 amino acids at either or at both the recited extremes.Especially particularly highly preferred are ranges plus or minus 1amino acid at either or at both extremes or the recited ranges with noadditions or deletions. Most highly preferred of all in this regard arefragments from about 150 to about 219 amino acids.

[0109] Among especially preferred fragments of the invention aretruncation mutants of h4-1BBSV receptor. Truncation mutants includeh4-1BBSV receptor polypeptides having the amino acid sequence of FIGS.1A-B (SEQ ID NO:2), or of variants or derivatives thereof, except fordeletion of a continuous series of residues (that is, a continuousregion, part or portion) that includes the amino terminus, or acontinuous series of residues that includes the carboxyl terminus or, asin double truncation mutants, deletion of two continuous series ofresidues, one including the amino terminus and one including thecarboxyl terminus. Fragments having the size ranges set out about alsoare preferred embodiments of truncation fragments, which are especiallypreferred among fragments generally.

[0110] Also preferred in this aspect of the invention are fragmentscharacterized by structural or functional attributes of h4-1BBSVreceptor. Preferred embodiments of the invention in this regard includefragments that comprise alpha-helix and alpha-helix forming regions(“alpha-regions”), beta-sheet and beta-sheet-forming regions(“beta-regions”), turn and turn-forming regions (“turn-regions”), coiland coil-forming regions (“coil-regions”), hydrophilic regions,hydrophobic regions, alpha amphipathic regions, beta amphipathicregions, flexible regions, surface-forming regions and high antigenicindex regions of h4-1BBSV receptor.

[0111] Certain preferred regions in these regards are set out in FIG. 3,and include, but are not limited to, regions of the aforementioned typesidentified by analysis of the amino acid sequence set out in FIGS. 1A-B(SEQ ID NO:2). As set out in FIG. 3, such preferred regions includeGarnier-Robson alpha-regions, beta-regions, turn-regions andcoil-regions, Chou-Fasman alpha-regions, beta-regions and turn-regions,Kyte-Doolittle hydrophilic regions and hydrophilic regions, Eisenbergalpha and beta amphipathic regions, Karplus-Schulz flexible regions,Emini surface-forming regions and Jameson-Wolf high antigenic indexregions.

[0112] Further preferred regions are those that mediate activities ofh4-1BBSV receptor. Most highly preferred in this regard are fragmentsthat have a chemical, biological or other activity of h4-1BBSV receptor,including those with a similar activity or an improved activity, or witha decreased undesirable activity. Highly preferred in this regard arefragments that contain regions that are homologs in sequence, or inposition, or in both sequence and to active regions of relatedpolypeptides, such as the related polypeptide set out in FIG. 2 (SEQ IDNO:9). Among particularly preferred fragments in these regards aretruncation mutants, as discussed above.

[0113] It will be appreciated that the invention also relates to, amongothers, polynucleotides encoding the aforementioned fragments,polynucleotides that hybridize to polynucleotides encoding thefragments, particularly those that hybridize under stringent conditions,and polynucleotides, such as PCR primers, for amplifying polynucleotidesthat encode the fragments. In these regards, preferred polynucleotidesare those that correspondent to the preferred fragments, as discussedabove.

[0114] Vectors, Host Cells, Expression

[0115] The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

[0116] Host cells can be genetically engineered to incorporatepolynucleotides and express polypeptides of the present invention. Forinstance, polynucleotides may be introduced into host cells using wellknown techniques of infection, transduction, transfection, transvectionand transformation. The polynucleotides may be introduced alone or withother polynucleotides. Such other polynucleotides may be introducedindependently, co-introduced or introduced joined to the polynucleotidesof the invention.

[0117] Thus, for instance, polynucleotides of the invention may betransfected into host cells with another, separate, polynucleotideencoding a selectable marker, using standard techniques forco-transfection and selection in, for instance, mammalian cells. In thiscase the polynucleotides generally will be stably incorporated into thehost cell genome.

[0118] Alternatively, the polynucleotides may be joined to a vectorcontaining a selectable marker for propagation in a host. The vectorconstruct may be introduced into host cells by the aforementionedtechniques. Generally, a plasmid vector is introduced as DNA in aprecipitate, such as a calcium phosphate precipitate, or in a complexwith a charged lipid. Electroporation also may be used to introducepolynucleotides into a host. If the vector is a virus, it may bepackaged in vitro or introduced into a packaging cell and the packagedvirus may be transduced into cells. A wide variety of techniquessuitable for making polynucleotides and for introducing polynucleotidesinto cells in accordance with this aspect of the invention are wellknown and routine to those of skill in the art. Such techniques arereviewed at length in Sambrook et al. cited above, which is illustrativeof the many laboratory manuals that detail these techniques.

[0119] In accordance with this aspect of the invention the vector maybe, for example, a plasmid vector, a single or double-stranded phagevector, a single or double-stranded RNA or DNA viral vector. Suchvectors may be introduced into cells as polynucleotides, preferably DNA,by well known techniques for introducing DNA and RNA into cells. Thevectors, in the case of phage and viral vectors also may be andpreferably are introduced into cells as packaged or encapsidated virusby well known techniques for infection and transduction. Viral vectorsmay be replication competent or replication defective. In the lattercase viral propagation generally will occur only in complementing hostcells.

[0120] Preferred among vectors, in certain respects, are those forexpression of polynucleotides and polypeptides of the present invention.Generally, such vectors comprise cis-acting control regions effectivefor expression in a host operatively linked to the polynucleotide to beexpressed. Appropriate trans-acting factors either are supplied by thehost, supplied by a complementing vector or supplied by the vectoritself upon introduction into the host.

[0121] In certain preferred embodiments in this regard, the vectorsprovide for specific expression. Such specific expression may beinducible expression or expression only in certain types of cells orboth inducible and cell-specific. Particularly preferred among induciblevectors are vectors that can be induced for expression by environmentalfactors that are easy to manipulate, such as temperature and nutrientadditives. A variety of vectors suitable to this aspect of theinvention, including constitutive and inducible expression vectors foruse in prokaryotic and eukaryotic hosts, are well known and employedroutinely by those of skill in the art.

[0122] The engineered host cells can be cultured in conventionalnutrient media, which may be modified as appropriate for, inter alia,activating promoters, selecting transformants or amplifying genes.Culture conditions, such as temperature, pH and the like, previouslyused with the host cell selected for expression generally will besuitable for expression of polypeptides of the present invention as willbe apparent to those of skill in the art.

[0123] A great variety of expression vectors can be used to express apolypeptide of the invention. Such vectors include chromosomal, episomaland virus-derived vectors e.g., vectors derived from bacterial plasmids,from bacteriophage, from yeast episomes, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids, all may be used for expression inaccordance with this aspect of the present invention. Generally, anyvector suitable to maintain, propagate or express polynucleotides toexpress a polypeptide in a host may be used for expression in thisregard.

[0124] The appropriate DNA sequence may be inserted into the vector byany of a variety of well-known and routine techniques. In general, a DNAsequence for expression is joined to an expression vector by cleavingthe DNA sequence and the expression vector with one or more restrictionendonucleases and then joining the restriction fragments together usingT4 DNA ligase. Procedures for restriction and ligation that can be usedto this end are well known and routine to those of skill. Suitableprocedures in this regard, and for constructing expression vectors usingalternative techniques, which also are well known and routine to thoseskill, are set forth in great detail in Sambrook et al. cited elsewhereherein.

[0125] The DNA sequence in the expression vector is operatively linkedto appropriate expression control sequence(s), including, for instance,a promoter to direct mRNA transcription. Representatives of suchpromoters include the phage lambda PL promoter, the E. coli lac, trp andtac promoters, the SV40 early and late promoters and promoters ofretroviral LTRs, to name just a few of the well-known promoters. It willbe understood that numerous promoters not mentioned are suitable for usein this aspect of the invention are well known and readily may beemployed by those of skill in the manner illustrated by the discussionand the examples herein.

[0126] In general, expression constructs will contain sites fortranscription initiation and termination, and, in the transcribedregion, a ribosome binding site for translation. The coding portion ofthe mature transcripts expressed by the constructs will include atranslation initiating AUG at the beginning and a termination codonappropriately positioned at the end of the polypeptide to be translated.

[0127] In addition, the constructs may contain control regions thatregulate as well as engender expression. Generally, in accordance withmany commonly practiced procedures, such regions will operate bycontrolling transcription, such as repressor binding sites andenhancers, among others.

[0128] Vectors for propagation and expression generally will includeselectable markers. Such markers also may be suitable for amplificationor the vectors may contain additional markers for this purpose. In thisregard, the expression vectors preferably contain one or more selectablemarker genes to provide a phenotypic trait for selection of transformedhost cells. Preferred markers include dihydrofolate reductase orneomycin resistance for eukaryotic cell culture, and tetracycline orampicillin resistance genes for culturing E. coli and other bacteria.

[0129] The vector containing the appropriate DNA sequence as describedelsewhere herein, as well as an appropriate promoter, and otherappropriate control sequences, may be introduced into an appropriatehost using a variety of well known techniques suitable to expressiontherein of a desired polypeptide. Representative examples of appropriatehosts include bacterial cells, such as E. coli, Streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells; insectcells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells suchas CHO, COS and Bowes melanoma cells; and plant cells. Hosts for of agreat variety of expression constructs are well known, and those ofskill will be enabled by the present disclosure readily to select a hostfor expressing a polypeptides in accordance with this aspect of thepresent invention.

[0130] More particularly, the present invention also includesrecombinant constructs, such as expression constructs, comprising one ormore of the sequences described above. The constructs comprise a vector,such as a plasmid or viral vector, into which such a sequence of theinvention has been inserted. The sequence may be inserted in a forwardor reverse orientation. In certain preferred embodiments in this regard,the construct further comprises regulatory sequences, including, forexample, a promoter, operably linked to the sequence. Large numbers ofsuitable vectors and promoters are known to those of skill in the art,and there are many commercially available vectors suitable for use inthe present invention.

[0131] The following vectors, which are commercially available, areprovided by way of example. Among vectors preferred for use in bacteriaare pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors,Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A,available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540,pRIT5 available from Pharmacia. Among preferred eukaryotic vectors arePWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; andpSVK3, PBPV, PMSG and PSVL available from Pharmacia. These vectors arelisted solely by way of illustration of the many commercially availableand well known vectors that are available to those of skill in the artfor use in accordance with this aspect of the present invention. It willbe appreciated that any other plasmid or vector suitable for, forexample, introduction, maintenance, propagation or expression of apolynucleotide or polypeptide of the invention in a host may be used inthis aspect of the invention.

[0132] Promoter regions can be selected from any desired gene usingvectors that contain a reporter transcription unit lacking a promoterregion, such as a chloramphenicol acetyl transferase (“cat”)transcription unit, downstream of restriction site or sites forintroducing a candidate promoter fragment; i.e., a fragment that maycontain a promoter. As is well known, introduction into the vector of apromoter-containing fragment at the restriction site upstream of the catgene engenders production of CAT activity, which can be detected bystandard CAT assays. Vectors suitable to this end are well known andreadily available. Two such vectors are pKK232-8 and pCM7. Thus,promoters for expression of polynucleotides of the present inventioninclude not only well known and readily available promoters, but alsopromoters that readily may be obtained by the foregoing technique, usinga reporter gene.

[0133] Among known bacterial promoters suitable for expression ofpolynucleotides and polypeptides in accordance with the presentinvention are the E. coli lacI and lacZ and promoters, the T3 and T7promoters, the gpt promoter, the lambda PR, PL promoters and the trppromoter. Among known eukaryotic promoters suitable in this regard arethe CMV immediate early promoter, the HSV thymidine kinase promoter, theearly and late SV40 promoters, the promoters of retroviral LTRs, such asthose of the Rous sarcoma virus (“RSV”), and metallothionein promoters,such as the mouse metallothionein-I promoter.

[0134] Selection of appropriate vectors and promoters for expression ina host cell is a well known procedure and the requisite techniques forexpression vector construction, introduction of the vector into the hostand expression in the host are routine skills in the art.

[0135] The present invention also relates to host cells containing theabove-described constructs discussed above. The host cell can be ahigher eukaryotic cell, such as a mammalian cell, or a lower eukaryoticcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell.

[0136] Introduction of the construct into the host cell can be effectedby calcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al. BASIC METHODS IN MOLECULARBIOLOGY, (1986).

[0137] Constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

[0138] Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook et al.,MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989).

[0139] Generally, recombinant expression vectors will include origins ofreplication, a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence, and a selectablemarker to permit isolation of vector containing cells after exposure tothe vector. Among suitable promoters are those derived from the genesthat encode glycolytic enzymes such as 3-phosphoglycerate kinase(“PGK”), a-factor, acid phosphatase, and heat shock proteins, amongothers. Selectable markers include the ampicillin resistance gene of E.coli and the trp1 gene of S. cerevisiae.

[0140] Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes may be increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act to increase transcriptionalactivity of a promoter in a given host cell-type. Examples of enhancersinclude the SV40 enhancer, which is located on the late side of thereplication origin at bp 100 to 270, the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

[0141] Polynucleotides of the invention, encoding the heterologousstructural sequence of a polypeptide of the invention generally will beinserted into the vector using standard techniques so that it isoperably linked to the promoter for expression. The polynucleotide willbe positioned so that the transcription start site is locatedappropriately 5′ to a ribosome binding site. The ribosome binding sitewill be 5′ to the AUG that initiates translation of the polypeptide tobe expressed. Generally, there will be no other open reading frames thatbegin with an initiation codon, usually AUG, and lie between theribosome binding site and the initiating AUG. Also, generally, therewill be a translation stop codon at the end of the polypeptide and therewill be a polyadenylation signal and a transcription termination signalappropriately disposed at the 3′ end of the transcribed region.

[0142] For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. The signals may beendogenous to the polypeptide or they may be heterologous signals.

[0143] The polypeptide may be expressed in a modified form, such as afusion protein, and may include not only secretion signals but alsoadditional heterologous functional regions. Thus, for instance, a regionof additional amino acids, particularly charged amino acids, may beadded to the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification or during subsequenthandling and storage. Also, region also may be added to the polypeptideto facilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stabilityand to facilitate purification, among others, are familiar and routinetechniques in the art.

[0144] Suitable prokaryotic hosts for propagation, maintenance orexpression of polynucleotides and polypeptides in accordance with theinvention include Escherichia coli, Bacillus subtilis and Salmonellatyphimurium. Various species of Pseudomonas, Streptomyces, andStaphylococcus are suitable hosts in this regard. Moreover, many otherhosts also known to those of skill may be employed in this regard.

[0145] As a representative but non-limiting example, useful expressionvectors for bacterial use can comprise a selectable marker and bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of the well known cloning vector pBR322(ATCC 37017). Such commercial vectors include, for example, pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec,Madison, Wis., USA). These pBR322 “backbone” sections are combined withan appropriate promoter and the structural sequence to be expressed.

[0146] Following transformation of a suitable host strain and growth ofthe host strain to an appropriate cell density, where the selectedpromoter is inducible it is induced by appropriate means (e.g.,temperature shift or exposure to chemical inducer) and cells arecultured for an additional period.

[0147] Cells typically then are harvested by centrifugation, disruptedby physical or chemical means, and the resulting crude extract retainedfor further purification.

[0148] Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents, suchmethods are well know to those skilled in the art.

[0149] Various mammalian cell culture systems can be ¹ employed forexpression, as well. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblast, described in Gluzman etal., Cell 23: 175 (1981). Other cell lines capable of expressing acompatible vector include for example, the C127, 3T3, CHO, HeLa, humankidney 293 and BHK cell lines.

[0150] Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation sites, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnon-transcribed sequences that are necessary for expression. In certainpreferred embodiments in this regard DNA sequences derived from the SV40splice sites, and the SV40 polyadenylation sites are used for requirednon-transcribed genetic elements of these types.

[0151] The h4-1BBSV receptor polypeptide can be recovered and purifiedfrom recombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.Well known techniques for refolding protein may be employed toregenerate active conformation when the polypeptide is denatured duringisolation and or purification.

[0152] Polypeptides of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue,in some cases as a result of host-mediated processes.

[0153] h4-1BBSV receptor polynucleotides and polypeptides may be used inaccordance with the present invention for a variety of applications,particularly those that make use of the chemical and biologicalproperties h4-1BBSV receptor. Among these are applications in treatmentof tumors, resistance to parasites, bacteria and viruses, to induceproliferation of endothelial cells and certain hematopoietic cells, totreat restenosis and to prevent certain autoimmune diseases afterstimulation of an h4-1BBSV receptor by an agonist. Additionalapplications relate to diagnosis and to treatment of disorders of cells,tissues and organisms. These aspects of the invention are illustratedfurther by the following discussion.

[0154] Polynucleotide Assays

[0155] This invention is also related to the use of the h4-1BBSVreceptor polynucleotides to detect complementary polynucleotides suchas, for example, as a diagnostic reagent. Detection of a mutated form ofh4-1BBSV receptor associated with a dysfunction will provide adiagnostic tool that can add or define a diagnosis of a disease orsusceptibility to a disease which results from under-expressionover-expression or altered expression of h4-1BBSV receptor or a solubleform thereof, such as, for example, tumors, cytotoxicity, viralinfection, autoimmune disease, AIDS and graft-host rejection.

[0156] Individuals carrying mutations in the h4-1BBSV receptor gene maybe detected at the DNA level by a variety of techniques. Nucleic acidsfor diagnosis may be obtained from a patient's cells, such as fromblood, urine, saliva, tissue biopsy and autopsy material. The genomicDNA may be used directly for detection or may be amplified enzymaticallyby using PCR prior to analysis. (Saiki et al., Nature, 324: 163-166(1986)). RNA or cDNA may also be used in the same ways. As an example,PCR primers complementary to the nucleic acid encoding h4-1BBSV receptorcan be used to identify and analyze h4-1BBSV receptor expression andmutations. For example, deletions and insertions can be detected by achange in size of the amplified product in comparison to the normalgenotype. Point mutations can be identified by hybridizing amplified DNAto radiolabeled h4-1BBSV receptor RNA or alternatively, radiolabeledh4-1BBSV receptor antisense DNA sequences. Perfectly matched sequencescan be distinguished from mismatched duplexes by RNase A digestion or bydifferences in melting temperatures.

[0157] Sequence differences between a reference gene and genes havingmutations also may be revealed by direct DNA sequencing. In addition,cloned DNA segments may be employed as probes to detect specific DNAsegments. The sensitivity of such methods can be greatly enhanced byappropriate use of PCR or another amplification method. For example, asequencing primer is used with double-stranded PCR product or asingle-stranded template molecule generated by a modified PCR. Thesequence determination is performed by conventional procedures withradiolabeled nucleotide or by automatic sequencing procedures withfluorescent-tags.

[0158] Genetic testing based on DNA sequence differences may be achievedby detection of alteration in electrophoretic mobility of DNA fragmentsin gels, with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230: 1242 (1985)).

[0159] Sequence changes at specific locations also may be revealed bynuclease protection assays, such as RNase and S1 protection or thechemical cleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci.,USA, 85: 4397-4401 (1985)).

[0160] Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,restriction fragment length polymorphisms (“RFLP”) and Southern blottingof genomic DNA.

[0161] In addition to more conventional gel-electrophoresis and DNAsequencing, mutations also can be detected by in situ analysis.

[0162] Chromosome Assays

[0163] The sequences of the present invention are also valuable forchromosome identification. The sequence is specifically targeted to andcan hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome. Few chromosome marking reagents based on actualsequence data (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

[0164] In certain preferred embodiments in this regard, the cDNA hereindisclosed is used to clone genomic DNA of a h4-1BBSV receptor gene. Thiscan be accomplished using a variety of well known techniques andlibraries, which generally are available commercially. The genomic DNAthe is used for in situ chromosome mapping using well known techniquesfor this purpose. Typically, in accordance with routine procedures forchromosome mapping, some trial and error may be necessary to identify agenomic probe that gives a good in situ hybridization signal.

[0165] In some cases, in addition, sequences can be mapped tochromosomes by preparing PCR primers (preferably 15-25 bp) from thecDNA. Computer analysis of the 3′ untranslated region of the gene isused to rapidly select primers that do not span more than one exon inthe genomic DNA, thus complicating the amplification process. Theseprimers are then used for PCR screening of somatic cell hybridscontaining individual human chromosomes. Only those hybrids containingthe human gene corresponding to the primer will yield an amplifiedfragment.

[0166] PCR mapping of somatic cell hybrids is a rapid procedure forassigning a particular DNA to a particular chromosome. Using the presentinvention with the same oligonucleotide primers, sublocalization can beachieved with panels of fragments from specific chromosomes or pools oflarge genomic clones in an analogous manner. Other mapping strategiesthat can similarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

[0167] Fluorescence in situ hybridization (“FISH”) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 50 or 60. For a review of this technique, see Verma et al.,HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES, Pergamon Press, NewYork (1988).

[0168] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. Such data are found, for example, inV. McKusick, MENDELIAN INHERITANCE IN MAN, available on line throughJohns Hopkins University, Welch Medical Library. The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through linkage analysis (coinheritance ofphysically adjacent genes).

[0169] Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

[0170] With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

[0171] Polypeptide Assays

[0172] The present invention also relates to diagnostic assays such asquantitative and diagnostic assays for detecting levels of h4-1BBSVreceptor protein, or the soluble form thereof, in cells and tissues,including determination of normal and abnormal levels. Thus, forinstance, a diagnostic assay in accordance with the invention fordetecting over-expression of h4-1BBSV receptor, or soluble form thereof,compared to normal control tissue samples may be used to detect thepresence of tumors, for example. Assay techniques that can be used todetermine levels of a protein, such as an h4-1BBSV receptor protein ofthe present invention, or a soluble form thereof, in a sample derivedfrom a host are well-known to those of skill in the art. Such assaymethods include radioimmunoassays, competitive-binding assays, WesternBlot analysis and ELISA assays. Among these ELISAs frequently arepreferred. An ELISA assay initially comprises preparing an antibodyspecific to h4-1BBSV receptor, or soluble form, preferably a monoclonalantibody. In addition a reporter antibody generally is prepared whichbinds to the monoclonal antibody. The reporter antibody is attached adetectable reagent such as radioactive, fluorescent or enzymaticreagent, in this example horseradish peroxidase enzyme.

[0173] To carry out an ELISA assay, a sample is removed from a host andincubated on a solid support, e.g. a polystyrene dish, that binds theproteins in the sample. Any free protein binding sites on the dish arethen covered by incubating with a non-specific protein such as bovineserum albumin. Next, the monoclonal antibody is incubated in the dishduring which time the monoclonal antibodies attach to any h4-1BBSVreceptor proteins attached to the polystyrene dish. Unbound monoclonalantibody is washed out with buffer. The reporter antibody linked tohorseradish peroxidase is placed in the dish resulting in binding of thereporter antibody to any monoclonal antibody bound to h4-1BBSV receptor,or soluble receptor. Unattached reporter antibody is then washed out.Reagents for peroxidase activity, including a calorimetric substrate arethen added to the dish. Immobilized peroxidase, linked to h4-1BBSVreceptor through the primary and secondary antibodies, produces acolored reaction product. The amount of color developed in a given timeperiod indicates the amount of h4-1BBSV receptor protein, or solubleform, present in the sample. Quantitative results typically are obtainedby reference to a standard curve.

[0174] A competition assay may be employed wherein antibodies specificto h4-1BBSV receptor, or soluble form, attached to a solid support andlabeled h4-1BBSV receptor and a sample derived from the host are passedover the solid support and the amount of label detected attached to thesolid support can be correlated to a quantity of h4-1BBSV receptor inthe sample.

[0175] Antibodies

[0176] The polypeptides, their fragments or other derivatives, oranalogs thereof, or cells expressing them can be used as an immunogen toproduce antibodies thereto. These antibodies can be, for example,polyclonal or monoclonal antibodies. The present invention also includeschimeric, single chain, and humanized antibodies, as well as Fabfragments, or the product of an Fab expression library. Variousprocedures known in the art may be used for the production of suchantibodies and fragments.

[0177] Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

[0178] For preparation of monoclonal antibodies, any technique whichprovides antibodies produced by continuous cell line cultures can beused. Examples include the hybridoma technique (Kohler, G. and Milstein,C., Nature 256: 495-497 (1975), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., Immunology Today 4: 72 (1983) andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R.Liss, Inc. (1985).

[0179] Techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produce singlechain antibodies to immunogenic polypeptide products of this invention.Also, transgenic mice, or other organisms such as other mammals, may beused to express humanized antibodies to immunogenic polypeptide productsof this invention.

[0180] The above-described antibodies may be employed to isolate or toidentify clones expressing the polypeptide or to purify the polypeptideof the present invention by attachment of the antibody to a solidsupport for isolation and/or purification by affinity chromatography.

[0181] h4-1BBSV Receptor Binding Molecules and Assays

[0182] The present invention provides a method for determining whether aligand not known to be capable of binding to and h4-1BBSV receptor canbind to such receptor which comprises contacting a mammalian cell whichexpresses such a receptor with a ligand under conditions permittingbinding of ligands to the receptor, detecting the presence of a ligandwhich binds to the receptor and thereby determining whether the ligandbinds to the receptor. An example of such a method comprises contactinga mammalian cell comprising an isolated DNA molecule encoding theh4-1BBSV receptor with a plurality of candidate ligands, determiningthose ligands which bind to the receptor, and thereby identifyingligands which specifically interact with and bind to a receptor of thepresent invention.

[0183] Agonists and Antagonists—Assays and Molecules

[0184] The invention also provides a method of screening compounds toidentify those which enhance or block the action of h4-1BBSV receptor oncells. An agonist is a compound which increases the natural biologicalfunctions of h4-1BBSV receptor or which functions in a manner similar toh4-1BBSV, while antagonists decrease or eliminate such functions.

[0185] In general, such screening procedures involve providingappropriate cells which express the receptor polypeptide of the presentinvention on the surface thereof. Such cells include cells from mammals,yeast, Drosophila or E. coli. In particular, a polynucleotide encodingthe receptor of the present invention is employed to transfect cells tothereby express the receptor. The expressed receptor is then contactedwith a test compound to observe binding, stimulation or inhibition of afunctional response.

[0186] One such screening procedure involves the use of melanophoreswhich are transfected to express the receptor of the present invention.Such a screening technique is described in PCT WO 92/01810 publishedFeb. 6, 1992.

[0187] Thus, for example, such assay may be employed for screening for acompound which inhibits activation of the receptor polypeptide of thepresent invention by contacting the melanophore cells which encode thereceptor with both the receptor ligand and a compound to be screened.Inhibition of the signal generated by the ligand indicates that acompound is a potential antagonist for the receptor, i.e., inhibitsactivation of the receptor.

[0188] The screen may be employed for determining a compound whichactivates the receptor by contacting such cells with compounds to bescreened and determining whether such compound generates a signal, i.e.,activates the receptor.

[0189] Other screening techniques include the use of cells which expressthe receptor (for example, transfected CHO cells) in a system whichmeasures extracellular pH changes caused by receptor activation, forexample, as described in Science, volume 246, pages 181-296 (October1989). For example, compounds may be contacted with a cell whichexpresses the receptor polypeptide of the present invention and a secondmessenger response, e.g. signal transduction or pH changes, may bemeasured to determine whether the potential compound activates orinhibits the receptor.

[0190] Another such screening technique involves introducing RNAencoding the receptor into Xenopus oocytes to transiently express thereceptor. The receptor oocytes may then be contacted with the receptorligand and a compound to be screened, followed by detection ofinhibition or activation of a calcium signal in the case of screeningfor compounds which are thought to inhibit activation of the receptor.

[0191] Another screening technique involves expressing the receptor inwhich the receptor is linked to a phospholipase C or D. Asrepresentative examples of such cells, there may be mentionedendothelial cells, smooth muscle cells, embryonic kidney cells, etc. Thescreening may be accomplished as hereinabove described by detectingactivation of the receptor or inhibition of activation of the receptorfrom the phospholipase second signal.

[0192] Another method involves screening for compounds which inhibitactivation of the receptor polypeptide of the present inventionantagonists by determining inhibition of binding of labeled ligand tocells which have the receptor on the surface thereof. Such a methodinvolves transfecting a eukaryotic cell with DNA encoding the receptorsuch that the cell expresses the receptor on its surface and contactingthe cell with a compound in the presence of a labeled form of a knownligand. The ligand can be labeled, e.g., by radioactivity. The amount oflabeled ligand bound to the receptors is measured, e.g., by measuringradioactivity of the receptors. If the compound binds to the receptor asdetermined by a reduction of labeled ligand which binds to thereceptors, the binding of labeled ligand to the receptor is inhibited.

[0193] Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polypeptide of the inventionand thereby inhibit or extinguish its activity. Potential antagonistsalso may be small organic molecules, a peptide, a polypeptide such as aclosely related protein or antibody that binds the same sites on abinding molecule, such as a receptor molecule, without inducing h4-1BBSVreceptor-induced activities, thereby preventing the action of h4-1BBSVreceptor by excluding h4-1BBSV receptor from binding. Examples of smallmolecules include but are not limited to small organic molecules,peptides or peptide-like molecules.

[0194] Other potential antagonists include antisense molecules.Antisense technology can be used to control gene expression throughantisense DNA or RNA or through triple-helix formation. Antisensetechniques are discussed, for example, in—Okano, J. Neurochem. 56: 560(1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENEEXPRESSION, CRC Press, Boca Raton, Fla. (1988). Triple helix formationis discussed in, for instance Lee et al., Nucleic Acids Research 6: 3073(1979); Cooney et al., Science 241: 456 (1988); and Dervan et al.,Science 251: 1360 (1991). The methods are based on binding of apolynucleotide to a complementary DNA or RNA. For example, the 5′ codingportion of a polynucleotide that encodes the mature polypeptide of thepresent invention may be used to design an antisense RNA oligonucleotideof from about 10 to 40 base pairs in length. A DNA oligonucleotide isdesigned to be complementary to a region of the gene involved intranscription thereby preventing transcription and the production ofh4-1BBSV receptor. The antisense RNA oligonucleotide hybridizes to themRNA in vivo and blocks translation of the mRNA molecule into h4-1BBSVreceptor polypeptide. The oligonucleotides described above can also bedelivered to cells such that the antisense RNA or DNA may be expressedin vivo to inhibit production of h4-1BBSV receptor.

[0195] A soluble form of the receptor, e.g. a fragment of the receptor,may be employed to inhibit activation of the receptor by binding toh4-1BBSV receptor ligand and preventing the ligand from interacting withmembrane bound receptors.

[0196] The human 4-1BB receptor may also be employed as an antagonist.Antibodies specific to h4-1BBSV receptor polypeptide of the presentinvention are unique in that they may be used as both agonists andantagonists depending upon which portion, or epitope, of the receptorthey are specific to as shown in published PCT Application WO 94/09137,which is hereby incorporated by reference. Fusing the soluble h4-1BBSVreceptor to Fc or hinge regions of immunoglobulins will increase thehalf-life of h4-1BBSV in vivo and increase binding to ligand due tomultimerization.

[0197] The antagonists may be employed in a composition with apharmaceutically acceptable carrier, e.g., as hereinafter described.

[0198] The antagonists may be employed for instance to treat to preventseptic shock, inflammation, cerebral malaria, activation of the HIVvirus, graft rejection, bone resorption and cachexia.

[0199] The agonists may be employed to treat and/or prevent tumors,restenosis, cytotoxicity, bacterial and viral infection, deleteriouseffects of ionizing radiation, autoimmune disease, AIDS and graft-hostrejection, to regulate immune responses, wound healing and cellularproliferation.

[0200] Compositions

[0201] The invention also relates to compositions comprising thepolynucleotide or the polypeptides discussed above or the agonists orantagonists. Thus, the polypeptides of the present invention may beemployed in combination with a non-sterile or sterile carrier orcarriers for use with cells, tissues or organisms, such as apharmaceutical carrier suitable for administration to a subject. Suchcompositions comprise, for instance, a media additive or atherapeutically effective amount of a polypeptide of the invention and apharmaceutically acceptable carrier or excipient. Such carriers mayinclude, but are not limited to, saline, buffered saline, dextrose,water, glycerol, ethanol and combinations thereof. The formulationshould suit the mode of administration.

[0202] Kits

[0203] The invention further relates to pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, reflecting approval by theagency of the manufacture, use or sale of the product for humanadministration.

[0204] Administration

[0205] Polypeptides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

[0206] The pharmaceutical compositions may be administered in anyeffective, convenient manner including, for instance, administration bytopical, oral, anal, vaginal, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal or intradermal routes amongothers.

[0207] The pharmaceutical compositions generally are administered in anamount effective for treatment or prophylaxis of a specific indicationor indications. In general, the compositions are administered in anamount of at least about 10 μg/kg body weight. In most cases they willbe administered in an amount not in excess of about 8 mg/kg body weightper day. Preferably, in most cases, dose is from about 10 μg/kg to about1 mg/kg body weight, daily. It will be appreciated that optimum dosagewill be determined by standard methods for each treatment modality andindication, taking into account the indication, its severity, route ofadministration, complicating conditions and the like.

[0208] Gene therapy.

[0209] The h4-1BBSV receptor polynucleotides, soluble form of thereceptor polypeptides, agonists and antagonists that are polypeptidesmay be employed in accordance with the present invention by expressionof such polypeptides in vivo, in treatment modalities often referred toas “gene therapy.”

[0210] Thus, for example, cells from a patient may be engineered with apolynucleotide, such as a DNA or RNA, encoding a polypeptide ex vivo,and the engineered cells then can be provided to a patient to be treatedwith the polypeptide. For example, cells may be engineered ex vivo bythe use of a retroviral plasmid vector containing RNA encoding apolypeptide of the present invention. Such methods are well-known in theart and their use in the present invention will be apparent from theteachings herein.

[0211] Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by procedures known in the art. For example, apolynucleotide of the invention may be engineered for expression in areplication defective retroviral vector, as discussed above. Theretroviral expression construct then may be isolated and introduced intoa packaging cell is transduced with a retroviral plasmid vectorcontaining RNA encoding a polypeptide of the present invention such thatthe packaging cell now produces infectious viral particles containingthe gene of interest. These producer cells may be administered to apatient for engineering cells in vivo and expression of the polypeptidein vivo. These and other methods for administering a polypeptide of thepresent invention by such method should be apparent to those skilled inthe art from the teachings of the present invention.

[0212] Retroviruses from which the retroviral plasmid vectors hereinabove mentioned may be derived include, but are not limited to, MoloneyMurine Leukemia Virus, spleen necrosis virus, retroviruses such as RousSarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon apeleukemia virus, human immunodeficiency virus, adenovirus,Myeloproliferative Sarcoma Virus, and mammary tumor virus. In oneembodiment, the retroviral plasmid vector is derived from Moloney MurineLeukemia Virus.

[0213] Such vectors well include one or more promoters for expressingthe polypeptide. Suitable promoters which may be employed include, butare not limited to, the retroviral LTR; the SV40 promoter; and the humancytomegalovirus (CMV) promoter described in Miller et al., Biotechniques7: 980-990 (1989), or any other promoter (e.g., cellular promoters suchas eukaryotic cellular promoters including, but not limited to, thehistone, RNA polymerase III, and β-actin promoters) Other viralpromoters which may be employed include, but are not limited to,adenovirus promoters, thymidine kinase (TK) promoters, and B19parvovirus promoters. The selection of a suitable promoter will beapparent to those skilled in the art from the teachings containedherein.

[0214] The nucleic acid sequence encoding the polypeptide of the presentinvention will be placed under the control of a suitable promoter.Suitable promoters which may be employed include, but are not limitedto, adenoviral promoters, such as the adenoviral major late promoter; orheterologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRs hereinabove described); the S-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

[0215] The retroviral plasmid vector is employed to transduce packagingcell lines to form producer cell lines. Examples of packaging cellswhich may be transfected include, but are not limited to, the PE501,PA317, Y-2, Y-AM, PA12, T19-14X, VT-19-17-H2, YCRE, YCRIP, GP+E-86,GP+envAm12, and DAN cell lines as described in Miller, A., Human GeneTherapy 1: 5-14 (1990). The vector may be transduced into the packagingcells through any means known in the art. Such means include, but arenot limited to, electroporation, the use of liposomes, and CaPO4precipitation. In one alternative, the retroviral plasmid vector may beencapsulated into a liposome, or coupled to a lipid, and thenadministered to a host.

[0216] The producer cell line will generate infectious retroviral vectorparticles, which include the nucleic acid sequence(s) encoding thepolypeptides. Such retroviral vector particles then may be employed totransduce eukaryotic cells, either in vitro or in vivo. The transducedeukaryotic cells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

EXAMPLES

[0217] The present invention is further described by the followingexamples. The examples are provided solely to illustrate the inventionby reference to specific embodiments. These exemplification's, whileillustrating certain specific aspects of the invention, do not portraythe limitations or circumscribe the scope of the disclosed invention.

[0218] Certain terms used herein are explained in the foregoingglossary.

[0219] All examples were carried out using standard techniques, whichare well known and routine to those of skill in the art, except whereotherwise described in detail. Routine molecular biology techniques ofthe following examples can be carried out as described in standardlaboratory manuals, such as Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989), herein referred to as “Sambrook.”

[0220] All parts or amounts set out in the following examples are byweight, unless otherwise specified.

[0221] Unless otherwise stated size separation of fragments in theexamples below was carried out using standard techniques of agarose andpolyacrylamide gel electrophoresis (“PAGE”) in Sambrook and numerousother references such as, for instance, by Goeddel et al., Nucleic AcidsRes. 8: 4057 (1980).

[0222] Unless described otherwise, ligations were accomplished usingstandard buffers, incubation temperatures and times, approximatelyequimolar amounts of the DNA fragments to be ligated and approximately10 units of T4 DNA ligase (“ligase”) per 0.5 μg of DNA.

Example 1

[0223] Expression and Purification of Human h4-1BBSV SolubleExtracellular Domain Using Bacteria

[0224] The DNA sequence encoding h4-1BBSV receptor in the depositedpolynucleotide was amplified using PCR oligonucleotide primers specificto the amino acid carboxyl terminal sequence of the h4-1BBSV receptorprotein and to vector sequences 3′ to the gene. Additional nucleotidescontaining restriction sites to facilitate cloning were added to the 5′and 3′ sequences respectively.

[0225] The 5′ oligonucleotide primer had the sequence 5′CGCCCATGGGAGAGGACAAGATCA 3′ (SEQ ID NO:3) containing the underlined NcoIrestriction site, which encodes a start AUG, followed by 16 nucleotidesof the h4-1BBSV receptor coding sequence set out in FIGS. 1A-B (SEQ IDNO:1) after the signal peptide.

[0226] The 3′ primer had the sequence 5° CGCGGTACCTCACTG CGGAGAGTG 3′(SEQ ID NO:4) containing the underlined Asp718 restriction site followedby 15 nucleotides complementary to the last 12 nucleotides of theh4-1BBSV receptor coding sequence for extracellular domain, includingthe stop codon.

[0227] The restrictions sites were convenient to restriction enzymesites in the bacterial expression vectors pQE-70 which were used forbacterial expression in these examples. (Qiagen, Inc. 9259 Eton Avenue,Chatsworth, Calif., 91311). pQE-70 encodes ampicillin antibioticresistance (“Ampr”) and contains a bacterial origin of replication(“ori”), an IPTG inducible promoter, a ribosome binding site (“RBS”), a6-His tag and restriction enzyme sites.

[0228] The amplified h4-1BBSV receptor DNA and the vector pQE-70 bothwere digested with NcoI and Asp718 and the digested DNAs then wereligated together. Insertion of the h4-1BBSV receptor DNA into the pQE-70restricted vector placed the h4-1BBSV receptor coding region downstreamof and operably linked to the vector's IPTG-inducible promoter andin-frame with an initiating AUG appropriately positioned for translationof h4-1BBSV receptor.

[0229] The ligation mixture was transformed into competent E. coli cellsusing standard procedures. Such procedures are described in Sambrook etal., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strainM15/rep4, containing multiple copies of the plasmid pREP4, whichexpresses lac repressor and confers kanamycin resistance (“Kanr”), wasused in carrying out the illustrative example described here. Thisstrain, which is only one of many that are suitable for expressingh4-1BBSV receptor, is available commercially from Qiagen.

[0230] Transformants were identified by their ability to grow on LBplates in the presence of ampicillin. Plasmid DNA was isolated fromresistant colonies and the identity of the cloned DNA was confirmed byrestriction analysis.

[0231] Clones containing the desired constructs were grown overnight(“O/N”) in liquid culture in LB media supplemented with both ampicillin(100 ug/ml) and kanamycin (25 ug/ml).

[0232] The O/N culture was used to inoculate a large culture, at adilution of approximately 1:100 to 1:250. The cells were grown to anoptical density at 600 nm (“OD600”) of between 0.4 and 0.6.Isopropyl-B-D-thiogalactopyranoside (“IPTG”) was then added to a finalconcentration of 1 mM to induce transcription from lac repressorsensitive promoters, by inactivating the lacI repressor. Cellssubsequently were incubated further for 3 to 4 hours. Cells then wereharvested by centrifugation and disrupted, by standard methods.Inclusion bodies were purified from the disrupted cells using routinecollection techniques, and protein was solubilized from the inclusionbodies into 8M urea. The 8M urea solution containing the solubilizedprotein was passed over a PD-10 column in 2× phosphate buffered saline(“PBS”), thereby removing the urea, exchanging the buffer and refoldingthe protein. The protein was purified by a further step ofchromatography to remove endotoxin. Then, it was sterile filtered. Thesterile filtered protein preparation was stored in 2X PBS at aconcentration of 95 micrograms per mL.

[0233] Analysis of the preparation by standard methods of polyacrylamidegel electrophoresis revealed that the preparation contained about 90%monomer h4-1BBSV receptor having the expected molecular weight of,approximately, 14 kDa.

Example 2

[0234] Cloning and Expression of the Soluble Extracellular Domainh4-1BBSV Receptor in a Baculovirus Expression System

[0235] The cDNA sequence encoding the soluble extracellular domain ofh4-1BBSV receptor protein in the deposited clone is amplified using PCRoligonucleotide primers corresponding to the 5′ and 3′ sequences of thegene:

[0236] The 5′ primer has the sequence 5′ CGCCCCGGGGCCATCATGGGA

[0237] AACAGCTGT 3′ (SEQ ID NO:5) containing the underlined Sma Irestriction enzyme site followed by Kozak sequence and 15 bases of thesequence of h4-1BBSV receptor of FIGS. 1A-B (SEQ ID NO:1). Inserted intoan expression vector, as described below, the 5′ end of the amplifiedfragment encoding h4-1BBSV receptor provides an efficient signalpeptide. An efficient signal for initiation of translation in eukaryoticcells, as described by Kozak, M., J. Mol. Biol. 196: 947-950 (1987) isappropriately located in the primer portion of the construct. The 3′primer has the sequence 5° CGCGGTACCTCACTGCGGAGAGTG 3′ (SEQ ID NO:6)containing the underlined Asp718 restriction followed by nucleotidescomplementary to bp 562 to 573 of the h4-1BBSV receptor coding sequenceset out in FIGS. 1A-B (SEQ ID NO:1), including the stop codon.

[0238] The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with BamH1 and Asp718 and againis purified on a 1% agarose gel. This fragment is designated herein F2.

[0239] The vector pA2 is used to express the h4-1BBSV receptor proteinin the baculovirus expression system, using standard methods, such asthose described in Summers et al, A MANUAL OF METHODS FOR BACULOVIRUSVECTORS AND INSECT CELL CULTURE PROCEDURES, Texas AgriculturalExperimental Station Bulletin No. 1555 (1987). This expression vectorcontains the strong polyhedrin promoter of the Autographa californicanuclear polyhedrosis virus (AcMNPV) followed by convenient restrictionsites. For an easy selection of recombinant virus the beta-galactosidasegene from E.coli is inserted in the same orientation as the polyhedrinpromoter and is followed by the polyadenylation signal of the polyhedringene. The polyhedrin sequences are flanked at both sides by viralsequences for cell-mediated homologous recombination with wild-typeviral DNA to generate viable virus that express the clonedpolynucleotide.

[0240] Many other baculovirus vectors could be used in place of pA2,such as pAc373, pVL941 and pAcIM1 provided, as those of skill readilywill appreciate, that construction provides appropriately locatedsignals for transcription, translation, trafficking and the like, suchas an in-frame AUG and a signal peptide, as required. Such vectors aredescribed in Luckow et al., Virology 170: 31-39, among others.

[0241] The plasmid is digested with the restriction enzymes Sma I andAsp718 and then is dephosphorylated using calf intestinal phosphatase,using routine procedures known in the art. The DNA is then isolated froma 1% agarose gel using a commercially available kit (“Geneclean” BIO 101Inc., La Jolla, Ca.). This vector DNA is designated herein “V2”.

[0242] Fragment F2 and the dephosphorylated plasmid V2 are ligatedtogether with T4 DNA ligase. E. coli HB101 cells are transformed withligation mix and spread on culture plates. Bacteria are identified thatcontain the plasmid with the human h4-1BBSV receptor gene by digestingDNA from individual colonies using Sma I and Asp718 and then analyzingthe digestion product by gel electrophoresis. The sequence of the clonedfragment is confirmed by DNA sequencing. This plasmid is designatedherein pBach4-1BBSV receptor.

[0243] 5 μg of the plasmid pBach4-1BBSV receptor is co-transfected with1.0 μg of a commercially available linearized baculovirus DNA(“BaculoGold™ baculovirus DNA”, Pharmingen, San Diego, Calif.), usingthe lipofection method described by Felgner et al., Proc. Natl. Acad.Sci. USA 84: 7413-7417 (1987). 1 μg of BaculoGold™ virus DNA and 5 μg ofthe plasmid pBach4-1BBSV receptor are mixed in a sterile well of amicrotiter plate containing 50 μl of serum free Grace's medium (LifeTechnologies Inc., Gaithersburg, Md.). Afterwards 10 μl Lipofectin plus90 μl Grace's medium are added, mixed and incubated for 15 minutes atroom temperature. Then the transfection mixture is added drop-wise toSf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture platewith 1 ml Grace's medium without serum. The plate is rocked back andforth to mix the newly added solution. The plate is then incubated for 5hours at 27°C. After 5 hours the transfection solution is removed fromthe plate and 1 ml of Grace's insect medium supplemented with 10% fetalcalf serum is added. The plate is put back into an incubator andcultivation is continued at 27° C. for four days.

[0244] After four days the supernatant is collected and a plaque assayis performed, as described by Summers and Smith, cited above. An agarosegel with “Blue Gal” (Life Technologies Inc., Gaithersburg) is used toallow easy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. (A detailed description of a “plaqueassay” of this type can also be found in the user's guide for insectcell culture and baculovirology distributed by Life Technologies Inc.,Gaithersburg, page 9-10).

[0245] Four days after serial dilution, the virus is added to the cells.After appropriate incubation, blue stained plaques are picked with thetip of an Eppendorf pipette. The agar containing the recombinant virusesis then resuspended in an Eppendorf tube containing 200 μl of Grace'smedium. The agar is removed by a brief centrifugation and thesupernatant containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4° C. A clonecontaining properly inserted h4-1BBSV receptor is identified by DNAanalysis including restriction mapping and sequencing. This isdesignated herein as V-h4-1BBSV receptor.

[0246] Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbaculovirus V-h4-1BBSV receptor at a multiplicity of infection (“MOI”)of about 2 (about 1 to about 3). Six hours later the medium is removedand is replaced with SF900 II medium minus methionine and cysteine(available from Life Technologies Inc., Gaithersburg). 42 hours later, 5μCi of 35S-methionine and 5 μCi 35S cysteine (available from Amersham)are added. The cells are further incubated for 16 hours and then theyare harvested by centrifugation, lysed and the labeled proteins arevisualized by SDS-PAGE and autoradiography.

Example 3

[0247] Expression of Soluble Extracellular Domain h4-1BBSV Receptor inCOS Cells

[0248] The expression plasmid, h4-1BBSV receptor HA, is made by cloninga cDNA encoding h4-1BBSV receptor into the expression vector pcDNAI/Amp(which can be obtained from Invitrogen, Inc.).

[0249] The expression vector pcDNAI/amp contains: (1) an E.coli originof replication effective for propagation in E. coli and otherprokaryotic cell; (2) an ampicillin resistance gene for selection ofplasmid-containing prokaryotic cells; (3) an SV40 origin of replicationfor propagation in eukaryotic cells; (4) a CMV promoter, a polylinker,an SV40 intron, and a polyadenylation signal arranged so that a cDNAconveniently can be placed under expression control of the CMV promoterand operably linked to the SV40 intron and the polyadenylation signal bymeans of restriction sites in the polylinker.

[0250] A DNA fragment encoding the entire h4-1BBSV receptor precursorand a HA tag fused in frame to its 3′ end is cloned into the polylinkerregion of the vector so that recombinant protein expression is directedby the CMV promoter. The HA tag corresponds to an epitope derived fromthe influenza hemagglutinin protein described by Wilson et al., Cell 37:767 (1984). The fusion of the HA tag to the target protein allows easydetection of the recombinant protein with an antibody that recognizesthe HA epitope.

[0251] The plasmid construction strategy is as follows.

[0252] The h4-1BBSV receptor cDNA of the deposit clone is amplifiedusing primers that contained convenient restriction sites, much asdescribed above regarding the construction of expression vectors forexpression of h4-1BBSV receptor in E. coli and S. fugiperda.

[0253] To facilitate detection, purification and characterization of theexpressed h4-1BBSV receptor, one of the primers contains a hemagglutinintag (“HA tag”) as described above.

[0254] Suitable primers include that following, which are used in thisexample.

[0255] The 5′ primer, 5° CGCGGATCCACCATGGGAAACAGCTGT 3′ (SEQ ID NO:7)contains the underlined Bam HI site, an ATG start codon and 12 codonsthereafter.

[0256] The 3′ primer, containing the underlined Xba I site and bp 562 to573 of 3′ coding sequence (at the 3′ end) has the following sequence; 5′CGCTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTACTGCGGAGAGTG 3′ (SEQ ID NO:8),the hemagglutinin tag is shown in bold.

[0257] The PCR amplified DNA fragment and the vector, pcDNAI/Amp, aredigested with Bam HI and Xba I and then ligated. The ligation mixture istransformed into E. coli strain SURE (available from Stratagene CloningSystems, 11099 North Torrey Pines Road, La Jolla, Calif. 92037) thetransformed culture is plated on ampicillin media plates which then areincubated to allow growth of ampicillin resistant colonies. Plasmid DNAis isolated from resistant colonies and examined by restriction analysisand gel sizing for the presence of the h4-1BBSV receptor-encodingfragment.

[0258] For expression of recombinant h4-1BBSV receptor, COS cells aretransfected with an expression vector, as described above, usingDEAE-DEXTRAN, as described, for instance, in Sambrook et al., MOLECULARCLONING: A LABORATORY MANUAL, Cold Spring Laboratory Press, Cold SpringHarbor, New York (1989). Cells are incubated under conditions forexpression of h4-1BBSV receptor by the vector.

[0259] Expression of the h4-1BBSV receptor HA fusion protein is detectedby radiolabelling and immunoprecipitation, using methods described in,for example Harlow et al., ANTIBODIES: A LABORATORY MANUAL, 2nd Ed.;Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988). Tothis end, two days after transfection, the cells are labeled byincubation in media containing 35S-cysteine for 8 hours. The cells andthe media are collected, and the cells are washed and the lysed withdetergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 1%NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. citedabove. Proteins are precipitated from the cell lysate and from theculture media using an HA-specific monoclonal antibody. The precipitatedproteins then are analyzed by SDS-PAGE gels and autoradiography. Anexpression product of the expected size is seen in the cell lysate,which is not seen in negative controls.

Example 4

[0260] Tissue Distribution of h4-1BBSV Receptor Expression

[0261] Northern blot analysis is carried out to examine the levels ofexpression of h4-1BBSV receptor in human tissues, using methodsdescribed by, among others, Sambrook et al, cited above. Total cellularRNA samples are isolated with RNAzol™ B system (Biotecx Laboratories,Inc. 6023 South Loop East, Houston, Tex. 77033).

[0262] About 10 μg of Total RNA is isolated from tissue samples. The RNAis size resolved by electrophoresis through a 1% agarose gel understrongly denaturing conditions. RNA is blotted from the gel onto a nylonfilter, and the filter then is prepared for hybridization to adetectably labeled polynucleotide probe.

[0263] As a probe to detect mRNA that encodes h4-1BBSV receptor, theantisense strand of the coding region of the cDNA insert in thedeposited clone is labeled to a high specific activity. The cDNA islabeled by primer extension, using the Prime-It kit, available fromStratagene. The reaction is carried out using 50 ng of the cDNA,following the standard reaction protocol as recommended by the supplier.The labeled polynucleotide is purified away from other labeled reactioncomponents by column chromatography using a Select-G-50 column, obtainedfrom 5-Prime -3-Prime, Inc. of 5603 Arapahoe Road, Boulder, Colo. 80303.

[0264] The labeled probe is hybridized to the filter, at a concentrationof 1,000,000 cpm/ml, in a small volume of 7% SDS, 0.5 M NaPO4, pH 7.4 at65° C., overnight.

[0265] Thereafter the probe solution is drained and the filter is washedtwice at room temperature and twice at 60° C. with 0.5× SSC, 0.1% SDS.The filter then is dried and exposed to film at −70° C. overnight withan intensifying screen.

Example 5

[0266] Gene Therapeutic Expression of Human h4-1BBSV Receptor

[0267] Fibroblasts are obtained from a subject by skin biopsy. Theresulting tissue is placed in tissue-culture medium and separated intosmall pieces. Small chunks of the tissue are placed on a wet surface ofa tissue culture flask, approximately ten pieces are placed in eachflask. The flask is turned upside down, closed tight and left at roomtemperature overnight. After 24 hours at room temperature, the flask isinverted—the chunks of tissue remain fixed to the bottom of theflask—and fresh media is added (e.g., Ham's F12 media, with 10% FBS,penicillin and streptomycin). The tissue is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerges. The monolayer istrypsinized and scaled into larger flasks.

[0268] A vector for gene therapy is digested with restriction enzymesfor cloning a fragment to be expressed. The digested vector is treatedwith calf intestinal phosphatase to prevent self-ligation. Thedephosphorylated, linear vector is fractionated on an agarose gel andpurified.

[0269] h4-1BBSV cDNA capable of expressing active h4-1BBSV receptor, isisolated. The ends of the fragment are modified, if necessary, forcloning into the vector. For instance, 5′ overhanging may be treatedwith DNA polymerase to create blunt ends. 3′ overhanging ends may beremoved using S1 nuclease. Linkers may be ligated to blunt ends with T4DNA ligase.

[0270] Equal quantities of the Moloney murine leukemia virus linearbackbone and the h4-1BBSV receptor fragment are mixed together andjoined using T4 DNA ligase. The ligation mixture is used to transform E.Coli and the bacteria are then plated onto agar-containing kanamycin.Kanamycin phenotype and restriction analysis confirm that the vector hasthe properly inserted gene.

[0271] Packaging cells are grown in tissue culture to confluent densityin Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS),penicillin and streptomycin. The vector containing the h4-1BBSV receptorgene is introduced into the packaging cells by standard techniques.Infectious viral particles containing the h4-1BBSV receptor gene arecollected from the packaging cells, which now are called producer cells.

[0272] Fresh media is added to the producer cells, and after anappropriate incubation period media is harvested from the plates ofconfluent producer cells. The media, containing the infectious viralparticles, is filtered through a Millipore filter to remove detachedproducer cells. The filtered media then is used to infect fibroblastcells. Media is removed from a sub-confluent plate of fibroblasts andquickly replaced with the filtered media. Polybrene (Aldrich) may beincluded in the media to facilitate transduction. After appropriateincubation, the media is removed and replaced with fresh media. If thetiter of virus is high, then virtually all fibroblasts will be infectedand no selection is required. If the titer is low, then it is necessaryto use a retroviral vector that has a selectable marker, such as neo orhis, to select out transduced cells for expansion.

[0273] Engineered fibroblasts then may be injected into rats, eitheralone or after having been grown to confluence on microcarrier beads,such as cytodex 3 beads. The injected fibroblasts produce h4-1BBSVreceptor product, and the biological actions of the protein are conveyedto the host.

[0274] It will be clear that the invention may be practiced otherwisethan as particularly described in the foregoing description andexamples.

[0275] Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, are withinthe scope of the appended claims.

1 9 1 946 DNA Homo sapiens CDS (124)..(783) 1 gcacgaggga aagttctccggcagccctga gatctcaaga gtgacatttg tgagaccagc 60 taatttgatt aaaattctcttggaatcagc tttgctagta tcatacctgt gccagatttc 120 atc atg gga aac agc tgttac aac ata gta gcc act ctg ttg ctg gtc 168 Met Gly Asn Ser Cys Tyr AsnIle Val Ala Thr Leu Leu Leu Val -15 -10 -5 ctc aac ttt gag agg aca agatca ttg cag gat cct tgt agt aac tgc 216 Leu Asn Phe Glu Arg Thr Arg SerLeu Gln Asp Pro Cys Ser Asn Cys -1 1 5 10 cca gct ggt gtt ttc agg accagg aag gag tgt tcc tcc acc agc aat 264 Pro Ala Gly Val Phe Arg Thr ArgLys Glu Cys Ser Ser Thr Ser Asn 15 20 25 gca gag tgt gac tgc act cca gggttt cac tgc ctg ggg gca gga tgc 312 Ala Glu Cys Asp Cys Thr Pro Gly PheHis Cys Leu Gly Ala Gly Cys 30 35 40 45 agc atg tgt gaa cag gat tgt aaacaa ggt caa gaa ctg aca aaa aaa 360 Ser Met Cys Glu Gln Asp Cys Lys GlnGly Gln Glu Leu Thr Lys Lys 50 55 60 ggt tgt aaa gac tgt tgc ttt ggg acattt aac gat cag aaa cgt ggc 408 Gly Cys Lys Asp Cys Cys Phe Gly Thr PheAsn Asp Gln Lys Arg Gly 65 70 75 atc tgt cga ccc tgg aca aac tgt tct ttggat gga aag tct gtg ctt 456 Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu AspGly Lys Ser Val Leu 80 85 90 gtg aat ggg acg aag gag agg gac gtg gtc tgtgga cca tct tca gcc 504 Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys GlyPro Ser Ser Ala 95 100 105 gac ctc tct ccg gga gca tcc tct gtg acc ccgcct gcc cct gcg aga 552 Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro ProAla Pro Ala Arg 110 115 120 125 gag cca gga cac tct ccg cag atc atc tccttc ttt ctt gcg ctg acg 600 Glu Pro Gly His Ser Pro Gln Ile Ile Ser PhePhe Leu Ala Leu Thr 130 135 140 tcg act gcg ttg ctc ttc ctg ctg ttc ttcctc acg ctc cgt ttc tct 648 Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe LeuThr Leu Arg Phe Ser 145 150 155 gtt gtt aaa cgg ggc aga aag aaa ctc ctgtat ata ttc aaa caa cca 696 Val Val Lys Arg Gly Arg Lys Lys Leu Leu TyrIle Phe Lys Gln Pro 160 165 170 ttt atg aga cca gta caa act act caa gaggaa gat ggc tgt agc tgc 744 Phe Met Arg Pro Val Gln Thr Thr Gln Glu GluAsp Gly Cys Ser Cys 175 180 185 cga ttt cca gaa gaa gaa gaa gga gga tgtgaa ctg tgaaatggaa 790 Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu190 195 200 gtcaataggg ctgttgggac tttcttgaaa agaagcaagg aaatatgagtcatccgctat 850 cacagctttc aaaagcaaga acaacatcct acattatacc caggattcccccaacacacg 910 ttcttttctt aatgccaatg agtgggcctt taaaaa 946 2 219 PRTHomo sapiens 2 Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu LeuVal Leu -15 -10 -5 Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys SerAsn Cys Pro -1 1 5 10 Ala Gly Val Phe Arg Thr Arg Lys Glu Cys Ser SerThr Ser Asn Ala 15 20 25 30 Glu Cys Asp Cys Thr Pro Gly Phe His Cys LeuGly Ala Gly Cys Ser 35 40 45 Met Cys Glu Gln Asp Cys Lys Gln Gly Gln GluLeu Thr Lys Lys Gly 50 55 60 Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn AspGln Lys Arg Gly Ile 65 70 75 Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp GlyLys Ser Val Leu Val 80 85 90 Asn Gly Thr Lys Glu Arg Asp Val Val Cys GlyPro Ser Ser Ala Asp 95 100 105 110 Leu Ser Pro Gly Ala Ser Ser Val ThrPro Pro Ala Pro Ala Arg Glu 115 120 125 Pro Gly His Ser Pro Gln Ile IleSer Phe Phe Leu Ala Leu Thr Ser 130 135 140 Thr Ala Leu Leu Phe Leu LeuPhe Phe Leu Thr Leu Arg Phe Ser Val 145 150 155 Val Lys Arg Gly Arg LysLys Leu Leu Tyr Ile Phe Lys Gln Pro Phe 160 165 170 Met Arg Pro Val GlnThr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg 175 180 185 190 Phe Pro GluGlu Glu Glu Gly Gly Cys Glu Leu 195 200 3 24 DNA Artificial sequenceContains an NcoI restriction site 3 cgcccatggg agaggacaag atca 24 4 24DNA Artificial sequence Contains an Asp718 restriction site 4 cgcggtacctcactgcggag agtg 24 5 30 DNA Artificial sequence Contains a Sma Irestriction enzyme site followed by Kozak sequence 5 cgccccggggccatcatggg aaacagctgt 30 6 24 DNA Artificial sequence Contains an Asp718restriction site 6 cgcggtacct cactgcggag agtg 24 7 27 DNA Artificialsequence Contains a Bam HI site 7 cgcggatcca ccatgggaaa cagctgt 27 8 51DNA Artificial sequence Contains an Xba I site and a hemagglutinin tag 8cgctctagat caagcgtagt ctgggacgtc gtatgggtac tgcggagagt g 51 9 255 PRTHomo sapiens 9 Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu LeuVal Leu 1 5 10 15 Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys SerAsn Cys Pro 20 25 30 Ala Gly Thr Phe Cys Asp Asn Asn Arg Asn Gln Ile CysSer Pro Cys 35 40 45 Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg ThrCys Asp Ile 50 55 60 Cys Arg Gln Cys Lys Gly Val Phe Arg Thr Arg Lys GluCys Ser Ser 65 70 75 80 Thr Ser Asn Ala Glu Cys Asp Cys Thr Pro Gly PheHis Cys Leu Gly 85 90 95 Ala Gly Cys Ser Met Cys Glu Gln Asp Cys Lys GlnGly Gln Glu Leu 100 105 110 Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe GlyThr Phe Asn Asp Gln 115 120 125 Lys Arg Gly Ile Cys Arg Pro Trp Thr AsnCys Ser Leu Asp Gly Lys 130 135 140 Ser Val Leu Val Asn Gly Thr Lys GluArg Asp Val Val Cys Gly Pro 145 150 155 160 Ser Pro Ala Asp Leu Ser ProGly Ala Ser Ser Val Thr Pro Pro Ala 165 170 175 Pro Ala Arg Glu Pro GlyHis Ser Pro Gln Ile Ile Ser Phe Phe Leu 180 185 190 Ala Leu Thr Ser ThrAla Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu 195 200 205 Arg Phe Ser ValVal Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe 210 215 220 Lys Gln ProPhe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly 225 230 235 240 CysSer Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 245 250 255

What is claimed is:
 1. An isolated polynucleotide comprising apolynucleotide having at least a 90% identity to a member selected fromthe group consisting of: (a) a polynucleotide encoding a polypeptidecomprising amino acid 1 to amino acid 132 set forth in SEQ ID NO:2; (b)a polynucleotide encoding a polypeptide comprising amino acid 1 to aminoacid 201 set forth in SEQ ID NO:2; (c) a polynucleotide which iscomplementary to the polynucleotide of (a) or (b).
 2. The polynucleotideof claim 1 wherein the polynucleotide is DNA.
 3. The polynucleotide ofclaim 1 wherein the polynucleotide is RNA.
 4. The polynucleotide ofclaim 1 wherein the polynucleotide is genomic DNA.
 5. The polynucleotideof claim 2 which encodes a polypeptide comprising amino acid 1 to 132 ofSEQ ID NO:2.
 6. The polynucleotide of claim 2 which encodes apolypeptide comprising amino acid 1 to 201 of SEQ ID NO:2.
 7. Anisolated polynucleotide comprising a polynucleotide having at least a90% identity to a member selected from the group consisting of: (a) apolynucleotide which encodes a mature polypeptide having the amino acidsequence expressed by the human cDNA contained in the deposited clone;and (b) a polynucleotide which is complementary to the polynucleotide of(a).
 8. The polynucleotide of claim 1 comprising the sequence as setforth in SEQ ID NO:1 from nucleotide 1 to nucleotide
 947. 9. Thepolynucleotide of claim 1 comprising the sequence as set forth in SEQ IDNO:1 from nucleotide 178 to nucleotide
 573. 10. The polynucleotide ofclaim 1 comprising the sequence as set forth in SEQ ID NO:1 fromnucleotide 178 to nucleotide
 780. 11. A vector comprising the DNA ofclaim
 1. 12. A host cell comprising the vector of claim
 11. 13. Aprocess for producing a polypeptide comprising: expressing from the hostcell of claim 12 the polypeptide encoded by said DNA.
 14. A process forproducing a cell comprising: genetically engineering the cell with thevector of claim 11 to thereby express the polypeptide encoded by the DNAcontained in the vector.
 15. A polypeptide comprising a member selectedfrom the group consisting of: (a) a polypeptide having an amino acidsequence set forth in SEQ ID NO:2; (b) a polypeptide comprising aminoacid 1 to amino acid 132 as set forth in SEQ ID NO:2; (c) a polypeptidecomprising amino acid 1 to amino acid 201 as set forth in SEQ ID NO:2;and (d) a polypeptide which is at least 85% identical to the polypeptideof (a).
 16. The polypeptide of claim 15 wherein the polypeptidecomprises amino acid 1 to amino acid 132 of SEQ ID NO:2.
 17. A compoundwhich inhibits activation of the polypeptide of claim
 15. 18. A compoundwhich activates the polypeptide of claim
 15. 19. A method for thetreatment of a patient having need of h4-1BBSV receptor comprising:administering to the patient a therapeutically effective amount of thepolypeptide of claim 15 by providing to the patient DNA encoding saidpolypeptide and expressing said polypeptide in vivo.
 20. A method forthe treatment of a patient having need to inhibit h4-1BBSV receptorcomprising: administering to the patient a therapeutically effectiveamount of the compound of claim
 17. 21. A process for diagnosing adisease or a susceptibility to a disease related to an under-expressionof the polypeptide of claim 15, comprising determining a mutation in anucleic acid sequence encoding said polypeptide.
 22. A diagnosticprocess, comprising analyzing for the presence of the polypeptide ofclaim 15 in a sample derived from a host.
 23. A method for identifyingagonists and antagonists to the polypeptide of claim 15 comprising:combining the polypeptide, a ligand known to bind to the polypeptide,and a compound to be screened under conditions where the ligand binds tothe polypeptide and determining whether the compound inhibits orenhances a signal generated by the polypeptide.
 24. The polypeptide ofclaim 15 wherein the polypeptide is a soluble fragment of thepolypeptide and is capable of binding a ligand for the receptor.
 25. Adiagnostic process, comprising analyzing for the presence of thepolypeptide of claim 24 in a sample derived from a host.